Experimental Studies in Equine Infectious Anemia 9781512815610

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THE EXPERIMENTAL WORK ON WHICH THIS MONOGRAPH IS BASED WAS PERFORMED AT THE SCHOOL OF V E T E R I N A R Y M E D I C I N E , U N I V E R S I T Y OF UNDER THE SPONSORSHIP OF THE GRAYSON

FOUNDATION

PENNSYLVANIA

EXPERIMENTAL STUDIES IN

EQUINE INFECTIOUS

ANEMIA

MIKLÓS N. DREGUSS, M.D. ASSOCIATE P R O F E S S O R O F V I R O L O G Y UNIVERSITY OF PENNSYLVANIA

and

LOUISE S. LOMBARD, D.V.M., Ph.D. R E S E A R C H ASSOCIATE IN P A T H O L O G Y UNIVERSITY OF PENNSYLVANIA

With 2 8 Illustrations and 1 6 Tables

UNIVERSITY PHILADELPHIA

OF

PENNSYLVANIA

PRESS 19 5 4

Copyright 1954 UNIVERSITY OF PENNSYLVANIA

Manufactured

in the United States of

PRESS

America

Library of Congress Catalog Card Number: 54-11539

Published in Great Britain, India, and Pakistan by Geoffrey Cumberlege: Oxford University Press London, Bombay, and Karachi

This

monograph

in gratitude

is

dedicated

to the memory

of

RAYMOND ALEXANDER KELSER (1892-1952) D E A N OF THE S C H O O L OF V E T E R I N A R Y M E D I C I N E UNIVERSITY OF PENNSYLVANIA

who was responsible of equine infectious

for initiating

the experimental

anemia at the University of

and who skillfully

guided and supervised

study

Pennsylvania

that work

NOTE BY THE PUBLISHER Dr. Miklös N. Dreguss received the degree of Doctor of Medicine from the University of Debrecen, Hungary, in 1928. After doing postgraduate research work at the University of Vienna in Austria, he became Assistant Professor of Medical Chemistry at the University of Debrecen. Following this, he did research work in the Children's Hospital of the same University. In 1934 he joined the staff of the National Institute of Health in Budapest, Hungary, and worked there, with interruptions, until 1947. In 1936-1937 he was engaged—under the tenure of a Rockefeller Foundation fellowship—in experimental work on influenza at the Rockefeller Institute, New York City, and at the National Institute for Medical Research in London, England. From 1939 on he was Head of the Virus Research Division of his home institute. After the war he continued his studies on virus diseases at the State Serum Institute in Copenhagen, Denmark. In 1948 he ira.v called to the University of Pennsylvania to participate—under Dean Kelser's guidance—in experimental work on infectious anemia. Dr. Dreguss has published over forty papers in American, Hungarian, German, and Swiss scientific journals, first in the field of biochemistry, and later in the field of various virus diseases (influenza, measles, poliomyelitis, typhus fever, primary atypical pneumonia, and equine anemia). Three times he received fellowships to do research work abroad, and lie was given the Balassa Award and the Högyes Prize by Medical Societies of his native country.

Dr. Louise S. Lombard received her degree of at Kansas State Veterinary College in 1944, and the at the University of Wisconsin in 1950. She taught Philadelphia, before she became associated in 1951 infectious anemia at the University of Pennsylvania. papers in the fields of sterility in cattle and cancer

Doctor of Veterinary Medicine degree of Doctor of Philosophy at Woman's Medical College in with the group studying equine Dr. Lombard has published research.

FOREWORD

A n intensive study of equine infectious anemia has been conducted at the School of Veterinary Medicine, University of Pennsylvania, under the sponsorship of the Grayson Foundation during the six-year period 19481953. This work was initiated by the late Dr. Raymond A. Kelser and had his direct guidance during most of the period. Active direction and execution of the laboratory aspects of the investigation were largely the responsibility of Dr. Miklos N. Dreguss, assisted for varying periods of time while the work was in progress by Dr. Werner Leemann, Dr. Arnold R. Gilman, Dr. Louise S. Lombard, and Miss Jane F. Shirer. The group applied in their experimental investigations the newer techniques of virology in attempts of adapting the virus to growth in culture and experimental animals, and in developing a more adequate method of accurate laboratory diagnosis. In addition, the research work included the study of the pathology of the disease and its blood picture for clarifying the mechanism of the infectious process. Part of the results of these researches have already been published in various scientific journals. In carrying out such a diverse attack on equine infectious anemia, an extensive amount of the large literature on the subject had to be reviewed, and Dr. Dreguss became impressed by how scattered and far-flung and frequently poorly accessible this literature was. It seemed to Dean Kelser and Dr. Dreguss that a monograph on the subject, drawing together all of the pertinent information within one publication, would serve a very useful purpose for those interested in either the laboratory or clinical aspects of the disease. The Grayson Foundation heartily concurred in this view and asked Dr. Dreguss to prepare, in cooperation with Dr. Lombard, such a monograph. The arduous task of reading and critically reviewing the hundreds of papers on the subject was carried out while the actual experimental work with equine infectious anemia was in progress at the University of Pennsylvania. The even more arduous task of converting this mass of

literature into readable chapters of a monograph has been conducted since the actual experimental work was completed at the University's old Bolton Farm near Bristol, Pennsylvania. The monograph includes not only the work of investigators of equine infectious anemia from all parts of the world, but both the published and unpublished original work of Dr. Dreguss and his group. It outlines in a very complete and concise manner our current understanding of the disease. The Grayson Foundation is grateful to Dr. Dreguss and Dr. Lombard for their willingness to undertake a task of the magnitude of this monograph and presents it in the hope that it will prove useful and valuable, not only to those interested in diseases of the horse and in animal husbandry but also to those seeking a clearer understanding of pathologic processes in virus diseases. The Rockefeller

Institute for Medical

New York, January

Research

1954

RICHARD E .

SHOPE,M.D.

Chairman, Scientific Advisory Committee of the Grayson Foundation

CONTENTS

Chapter

Page

1. History and Geographic Distribution

1

2.

8

Natural Transmission of the Infection

3. Symptomatology

17

4.

Clinical Diagnosis

22

5.

Properties of the Virus

26

6. Methods of Laboratory Diagnosis

31

7.

Hematology

. 4 8

8.

Hemagglutination

59

9.

Pathogenesis

71

10. Transmission to Laboratory Animals 11. Cultivation of the Virus in Chick Embryos

81 .

.

.

f

.

.

.102

12.

Immunization Experiments

Ill

13.

Histopathology

125

14. Therapy and Control

139

Concluding Remarks

145

Bibliography

149

Index

202

TABLES Table

Page

1. Comparative Temperature Figures in Different Scales . . . .

18

2. Complement-Fixation Test with Various Antigens

34

3. Sedimentation Rate of Erythrocytes in Infected Horses . . . .

39

4.

Hematologic Response of an Acute Case (Horse 2 9 )

.

5.

Hematologic Response of a Subacute Case (Horse 2 2 )

. . .

6.

Hematologic Response of Horse 33 (Subacute Case)

.

.

.

51

7.

Average of Hematologic Values in 15 Experimental Horses .

.

53

.

.

.

.

49 50

8. Hemagglutination Test in Experimentally Infected Horses . . .

62

9.

Hemagglutination Test Following Challenge Inoculation . . .

63

10.

Hemagglutination in Various Clinical Conditions

65

11.

Hemagglutination of Horse Red Blood Corpuscles

67

12.

Serum Gamma-globulin Level in Infected Horses

75

13.

Rabbits Inoculated with Infectious Horse Blood

86

14.

Hematologic Response of Rabbit 392 (19th Serial Transfer) .

15. Hematologic Findings in Passage Mice 16. Hematologic and Fever Response of Horse 25

.

89 97 117

CHARTS Chart

P'ige

1. Fever Response in Acute Infectious Anemia

73

2. Electrophoresis Pattern of Normal Horse Serum

77

3. Electrophoresis Pattern of Infectious Anemia Serum

. . . .

4. Fever Response of Rabbit 392 (19th Passage of Virus)

77

. . .

90

5. Fever Response of Horse 10 to Mouse Passage Material . . .

93

6. Fever Response of Horse 35

95

PLATES (Microphotographs) ?aSe

Plates

Color Plate: Fig. 1 to Fig. 6 Plates:

Fig. 7 to Fig. 22

78 127-135

CHAPTER 1

HISTORY

AND

GEOGRAPHIC

DISTRIBUTION

Nomenclature.—Since infectious anemia of horses was recognized and described as a clinical entity, many names have been used to designate the condition. At present the generally accepted term is Equine infectious anemia in English, or its equivalent in other languages. The following is a list of the more common synonyms used in various descriptions dealing with the disease: (English) Equine infectious anemia, Infectious anemia of horses, Swamp fever, Equine pernicious anemia, Pernicious anemia of horses, Equine relapsing fever (Watson), Equine malaria, American surra (Rutherford), Malarial fever of horses (Torrance), Manitoba disease, Mountain fever, No-name disease, Loin distemper, River bottom disease, Wyoming disease. (French) Anémie infectieuse du cheval, Typho-anémie infectieuse (Carré et Vallée), Hydrohémie du cheval (Lignée), Cachexie aqueuse du cheval (Lignée), Anhémie idiopathique du cheval (Anginiard), Anémie contagieuse du cheval, Anémie eqizootique des équidés, Anémie pernicieuse du cheval, Anémie pernicieuse progressive, Fièvre des malais, Maladie de Carré et Vallée, Maladie de Vallée, Malaria des chevaux. (German) Infektiöse Anämie der Pferde, Ansteckende Blutarmut der Pferde, Ansteckende Anämie der Pferde, Infektiöse Blutarmut der Pferde, Infektiöse Pferdeanämie, Perniziöse Anämie der Pferde, Progressive perniciöse Anämie der Pferde (Zschokke), Malaria des Pferdes, Rekurrierendes Fieber der Pferde (Liihrs), Wechselfieber des Pferdes, Sumpffieber, Augsburger Krankheit (Schotte), Augsburger Anämie (Ziegler), Virusanämie der Pferde (Krupski), Vallée-sche Krankheit. (Italian) Anemia infettiva del cavallo. (Spanish) Anemia infecciosa de los caballos, Anemia infecciosa de los equinos. {Dutch) Infectieuse Anaemia der Paarden. (Finnish) Näivetystautia. (Hungarian) Lovak fertözö kevésvérüsége, Fertözö vérszegénység. (Scandinavian languages) Infektiös anaemi hos hesten, Perniciös anämi hos (1)

2

EQUINE INFECTIOUS A N E M I A

hästar, Smittsom blodbrist hos hästar.

( S l a v o n i c languages)

Infekciozna

anemija, Infekcni anaemie koni, V i r o s n i anaemie, N i e d o k r w i t o s c zakazna koni. terms

Latin

suggested

(Fröhner),

equorum

for

Anaemia

international

use:

Anaemia

infectiosa

infectiosa equi, Septicaemia

intermittens

seu recidiva equi. T h e disease was first described in F r a n c e by L i g n é e ( 2 5 6 ) * in 1843 under the name of anhémie origin

connected

Charlier ( 6 8 )

in

Sanson

(378)

Ledru ( 2 4 1 )

manner

and D é n o c ( 7 8 )

while Anginiard ( 8 ) but

H e believed that it was of nutritional

du cheval.

some

with

the

artificial

diet

of

horses.

discussed the p r o b l e m in the same year,

concluded in 1859 that the disease was contagious, strongly

opposed

agreed with A n g i n i a r d .

this

concept.

Bouley

I n 1883 Zschokke

(44)

(498)

a clinically similar condition occurring in Switzerland; he tried, unsuccessfully, to transmit the disease.

and

reported though

In the same decade F r ö h n e r

(126)

and Ostertag ( 3 3 7 ) reported f r o m G e r m a n y cases of "pernicious a n e m i a " of horses that w e r e apparently infectious in nature.

I n the N e w

World

the first description of infectious anemia in the U n i t e d States b y W a t s o n (480)

appeared in

1896 under the name of

"equine

relapsing

fever,"

and in Canada by T o r r a n c e ( 4 5 7 ) in 1902 as " s w a m p f e v e r . " T h e identity of these diseases in Europe, the United States, and Canada, respectively, was later established. The

experiments

Veterinaire

d'A Ifort

to the systematic

of

Vallee

and

Carré

in France marked

study

of

the

(467,468) first

infectious anemia.

at

important

the

École

contribution

In their publications

in

1904 it was announced that they demonstrated experimentally the infectious nature of

the disease

causative agent. tional

research

problems

of

and have shown

that a

filterable

virus

was

the

Carré and V a l l é e submitted in a series of articles addidata

the

of

their

infection

work

in an attempt

(56,57,58).

Their

to c l a r i f y the

findings

were

basic

confirmed

and the clinical, diagnostic, and pathological features of the disease w e r e related in detail during the next decade by M a r e k ( 2 7 4 , 2 7 5 ) in H u n g a r y ; Ostertag

(338),

Hempel

(160),

G e r m a n y ; Francis and Marsteller and Schalk Wolbach

(470),

(451)

and

Scott

Lührs

(264),

(123), Mack

(395)

in the

and F r ö h n e r

United

(446)

in South A f r i c a ; Stadler

W i r t h ( 4 8 7 ) , Gerlach ( 1 3 2 ) , and JafiFé ( 1 8 8 )

(191)

(414)

in

Harris

States; T o d d

in C a n a d a ; the Japanese C o m m i s s i o n

T h e i l e r and K e h o e

(127)

( 2 7 1 ) , V a n Es,

and

in Japan; in

Sweden;

in Austria; and by many

others. O n the f o l l o w i n g pages a brief review will be presented of

reports

coming f r o m all parts of the g l o b e about the geographic distribution of infectious anemia and its prevalence all o v e r the w o r l d . * N u m b e r s in parentheses are references to the c o r r e s p o n d i n g listed in the bibliography at the end o f the m o n o g r a p h .

publications

as

1.

H I S T O R Y AND G E O G R A P H I C D I S T R I B U T I O N

3

Europe In 1843 infectious anemia was reported among horses in the M a r n e Valley of F r a n c e ( 6 8 , 7 8 , 2 5 6 ) . In 1906 it was enzootic ( 5 8 ) in AlsaceLorraine, in the entire Meuse Valley, in the departments of Aube, Ardennes, Còte d'Or, H a u t e M a r n e , M e u r t h e et Moselle, Meuse, and Seine et M a r n e . In Lorraine it was considered one of the most formidable of equine diseases attacking about half of the total cart-horse population ( 1 8 1 ) . L a m a r r e ( 2 3 4 ) analyzed the regional distribution of the disease in F r a n c e in 1933 when a serious outbreak occurred at Belfort. In 1935 a good review of the situation was published by Carré and Verge ( 6 1 ) . In Belgium the disease was first reported in 1906 by Béghin ( 3 4 ) , and Ries ( 3 7 2 ) believed that it was introduced into L u x e m b o u r g f r o m France. A s early as 1901 Kopeke ( 2 1 3 ) described an anemia of horses often ending fatally and occurring in epizootic f o r m during the late s u m m e r and a u t u m n months in the neighborhood of Metz, G e r m a n y . According to Hutyra, M a r e k and Manninger ( 1 8 1 ) infectious anemia first occurred in G e r m a n y in districts adjoining Lotharingia and later became known in the neighborhood of Augsburg in South Bavaria as "Augsburg disease." It was prevalent also in Hessen, East Prussia, and Schleswig. During and after the first World W a r the disease was conveyed to most of the remaining parts of G e r m a n y by infected cavalry horses ( 3 7 1 ) . This prompted a thorough scientific exploration of the problem by many investigators in several centers. In later years the severity of the disease decreased and it assumed a less acute and fatal form ( 1 8 1 ) . A review of its distribution in G e r m a n y f r o m 1918 to 1937 is given by Fortner ( 1 1 6 ) . M a r e k recognized the disease in Hungary in 1907 ( 1 8 0 , 2 7 4 ) and noted its spread during and after World W a r I when it assumed a more virulent type ( 1 8 1 ) . Later, Mócsy ( 2 9 3 - 3 0 0 ) published a series of reports. Its occurrence was recorded also in Austria ( 1 3 2 , 4 8 7 ) . Krai and his co-workers gave a comprehensive account of the disease in Czechoslovakia ( 2 2 2 ) . In Slovakia it was widespread after 1938 ( 4 8 2 ) ; in ten years between 1939 and 1948, 533 cases were officially reported ( 3 2 1 ) . Zwick ( 5 0 0 ) gave, in 1935, an account of its prevalence in Yugoslavia, while Zenkner and co-workers ( 4 9 5 ) reported it f r o m Poland. In Italy the disease was observed in L o m b a r d y ( 7 2 ) . Recently, A b b o n d a n z a ( 1 ) discussed the possibility of mistaking leptospirosis for infectious anemia in the Po Valley. In the opinion of Krupski and co-workers ( 2 2 4 , 2 2 6 ) true equine infectious anemia is comparatively rare in Switzerland, while an anemia of streptococcus origin is more c o m m o n . Steck ( 4 1 5 , 4 1 8 ) , however, maintains on the basis of clinical observations that the virus anemia is quite prevalent and that he had seen hundreds of cases during the period of 1932 to 1936. Notification

4

EQUINE INFECTIOUS A N E M I A

and isolation of diagnosed cases were made compulsory by the federal government in 1942. In northern Sweden the disease was first observed in 1895 by Ekvall ( 1 0 6 ) , and it was prevalent in central and northern parts of Sweden in 1932. Regulations by the state for its control were in effect in 1934. The morbidity in northern Sweden was listed at about 300 to 400 horses annually as related by Hjarre. In 1942 the State Veterinary Service officially diagnosed 128 cases in Sweden which number decreased to 31 in 1943. From the districts bordering on Sweden, the disease spread to Norway around 1909, but in 1934 only a few cases were observed in the latter country after a period of ten years during which strict control measures were employed ( 1 7 2 , 4 4 8 ) . Infectious anemia was noted about 1909 in Finland ( 1 8 1 ) ; Hallfors ( 1 4 8 ) , while checking thousands of horses close to the Swedish border, saw a number of chronic anemic cases. As recorded by Bartels ( 3 1 ) equine infectious anemia was unknown in Denmark until Niendahl diagnosed some cases in the district near Schleswig-Holstein. Since a law for the control of the disease was established in 1923, no case has been reported. Great Britain and the Iberian Peninsula are reportedly free of the malady ( 7 5 , 2 8 ) . A merica In western Canada the disease was first referred to near Winnipeg in the Province of Manitoba. Veterinarians settling in Manitoba were confronted, in 1881 and 1882, with equine anemia called "swamp fever" as related by Torrance ( 4 5 6 ) . It was demonstrated to be infectious by inoculation of blood from sick horses into a healthy animal ( 4 5 7 ) . The disease was, in 1902, prevalent among horses in a large area of the Province and in some parts of Northwest Territories; it was first identified in Saskatchewan in 1906. Todd and Wolbach ( 4 5 1 ) studied the problem of swamp fever experimentally and compared it with infectious anemia as reported from Europe and with equine anemia as described by several investigators in the United States. By 1942, the disease had spread west from Manitoba to the Rocky Mountains and east to Ontario and Quebec. In 1941, 121 cases were officially reported for Quebec while in 1942 the number increased to 218. Mitchell and associates ( 2 9 0 ) reported in 1944 an outbreak in the Ottawa Valley. Watson (480) in 1896 described a malady of horses in the United States under the name of "equine relapsing fever" that had first appeared about eight years previously in northern Wisconsin and which apparently was infectious anemia. According to Scott ( 3 9 6 ) infectious anemia made its appearance in Wyoming during the last decade of the 19th century and, in 1901, caused an extensive outbreak in that state. Brimhall

1.

H I S T O R Y AND G E O G R A P H I C D I S T R I B U T I O N

5

and associates ( 4 6 ) reported the condition in 1903 as observed in Minnesota. Möhler stated in the Bureau of Animal Industry's report for 1908 that the disease was prevalent in seven states ( 3 0 2 ) . According to Mack ( 2 7 1 ) , who made a careful inquiry into the problem of a fatal anemic disease of horses in Nevada, swamp fever was said in 1909 to exist in addition to Nevada, in Minnesota, Wyoming, Montana, North Dakota, Kansas, Nebraska, Colorado, and Texas. In several of these states investigative work was carried out in order to study the condition locally (123,271,395,444,470). The early history of infectious anemia in the United States and Canada is reviewed by Van Es, Harris and Schalk ( 4 7 0 ) in their monograph on swamp fever, and by Stein ( 4 2 5 ) . From 1916 to 1928 the prevalence of the disease in the States appeared to have decreased as judged on the basis of reports from veterinarians. Kelser ( 1 9 5 ) has recognized the nature of a severe outbreak in 1921 in brood mares at Fort Robinson, Nebraska, the infection having apparently been introduced by mares purchased in the vicinity. He also recorded the first occurrence of the disease in the New England states in 1935 ( 1 9 7 ) . According to a report published by the Bureau of Animal Industry in 1935, inquiries that were made after 1928 showed the disease to be existent in 17 states of the U. S. In 1941, Stein stated that it was apparently more prevalent than originally assumed: authentic accounts of the disease were received from 29 states and in 15 of these the diagnosis was confirmed by horse inoculation test ( 4 2 5 ) . Stein and Mott ( 4 3 5 ) further discussed the prevalence of equine infectious anemia in the United States on the occasion of a serious outbreak among thoroughbred horses at Rockingham Park, New Hampshire, in 1947, in which 80 horses were lost; this outbreak was reportedly traced to a race horse brought from Florida. They stated that during the past five decades the disease has been authentically reported from 34 of the 48 states, chiefly in the form of sporadic outbreaks. Small local outbreaks, showing little tendency to spread, occurred from time to time in certain areas in Idaho, Louisiana, Montana, Nevada, Oregon, Texas, and Wyoming, while the disease is enzootic in the Mississippi Delta where it exists in mules principally in chronic form. The Bureau of Animal Industry ( 4 6 2 ) stated that sporadic but unconfirmed equine infectious anemia cases were reported in ten states in 1951. Information concerning the prevalence of the disease in Mexico, Central and South America is meager, partly because of the difficulties in differentiating it from tropical diseases of the horse. Darling, writing from the Panama Canal Zone in 1910, made mention of a malady clinically corresponding to swamp fever ( 4 7 0 ) . In a description of equine anemia

6

EQUINE INFECTIOUS A N E M I A

as seen in Venezuela, Kubes (227) states that the disease was first observed in Brazil around 1905. He believed that of two clinical entities, "peste boba" and "derrengadera," the one named "peste boba" is in reality infectious anemia, and the other is an infection with Trypanosoma venezuelense. In Uruguay infectious anemia has been also reported and was studied experimentally in horses ( 6 2 ) . Asia There is no doubt that the disease is prevalent throughout the entire Eurasian continent. As early as 1909 many cases were recorded as occurring in low-lying swampy regions of Russia, particularly around the Rokitno and Pripet marshes ( 1 8 1 ) . Judging from the number of reports in Russian dealing briefly with problems and studies of the malady, without presenting details, it must be widespread in present-day U.S.S.R. There are early reports of the high incidence of the disease in Japan. In 1909 a special Commission was set up for the study and control of infectious anemia, and in 1914 it submitted a detailed account ( 1 9 1 ) of large numbers of field and experimental cases and also presented a draft of control measures for adoption. During the last three decades many papers have been published, most of them in Japanese, on experimental work performed in Japan by a number of investigators (Nagao, Nakamura, Ishii, Hirato, Kutii, and their co-workers). As reported by the Japanese Commission ( 1 9 1 ) , the first outbreaks of the disease had occurred in 1895 in the province of Hidaka on Hokkaido Island, in the prefecture of Aomori, and in Yokohama. The Japanese Livestock Bureau ( 1 9 0 ) listed the disease as known to exist in Java, French Indo-China, Formosa, the islands of Japan, Sakhalin, Korea and M a n churia. In Japan 2,302 cases were officially reported for 1949. In India equine surra (trypanosomiasis) and equine infectious anemia probably co-exist ( 5 8 2 ) , but apparently no serious effort has been made thus far toward a clear differentiation of the two diseases.

Africa and

Australia

In North Africa the disease was intensively studied by Balozet who used the African donkey for experimental transmission ( 1 3 ) . Accounts were given concerning the presence of infectious anemia in French Morocco (474,11), Tunisia ( 2 9 ) , and Eritrea ( 5 9 8 ) . The disease was first identified in South Africa by Theiler and Kehoe in 1913 ( 4 4 6 ) . De Kock ( 2 1 2 ) concluded, from a study of records, that it existed in South Africa even before 1904. He stated that there were isolated cases up to 1923, but that an extensive outbreak never occurred in horses; it was enzootic among donkeys of Natal, South Africa.

1.

H I S T O R Y AND G E O G R A P H I C D I S T R I B U T I O N

7

Infectious anemia has been recognized in Australia whence it spread, according to reports, to the Dutch East Indies ( 1 8 1 ) . Whittem relates that the disease was introduced to the island of New Britain in the Bismarck Archipelago by the Japanese invasion during World War II. However, it was soon eradicated and did not spread to continental Australia. *

In conclusion, it may be said that infectious anemia has been recognized and reported as being present on all continents and all geographic latitudes from the subpolar regions of Finland and Norway to the tropical climate such as that in Venezuela or Eritrea, with the exception only of Great Britain and the Iberian Peninsula (Balozet ( 2 8 ) ) . In a conference, held recently at the Office International des Epizooties in Paris ( 3 2 5 ) , prominent workers in this field from all over the world reported the situation in their respective countries about infectious anemia, the incidence of which is rising in some well defined regions of several countries. We may add in this connection that the mere absence of reports from a certain area does not necessarily exclude the presence of the malady among the equine population of that region, but may be attributed to lack of interest in the problem or, in the case of colonial territories and under-developed regions, the lack of competent investigators. Naturally, in countries in which the disease has been quite prevalent at one time or another, thus presenting an economic problem as well as a scientific challenge, more attention was paid to its surveillance and study than in places where its importance seemed negligible.

CHAPTER

2

NATURAL TRANSMISSION OF THE

INFECTION

Ever since it was demonstrated experimentally that the disease is infectious, investigators have been seeking to learn how infectious anemia is transmitted from one animal to another under natural conditions. Before discussing the modes of natural transmission, it is well to consider the susceptibility of horses to the virus, and to summarize briefly the views about the regional distribution of the disease. Observations as well as experiments revealed that the various equine species, including horses, mules, and donkeys, are susceptible to the infection in all age groups. In regions where the disease is enzootic, one may find some immune animals, an obvious explanation for this being that such animals have had a manifest or subclinical infection some time in the past and have gradually grown resistant to re-infection with the virus. In most instances, however, infection with the virus of infectious anemia does not confer immunity, a surprising fact that constantly baffled investigators studying the malady. Horses seemingly recovered f r o m an infection would usually produce a spontaneous flare-up of symptoms f r o m time to time (chronic cases)', some others would exhibit no symptoms for a long time, but their blood would retain infectiousness, as a rule, for the rest of their lives ( s y m p t o m l e s s virus carriers). In the latter group of seemingly normal horses, symptoms may recur after long quiescent periods without any apparent reason, or may be provoked by various methods, including re-infcction with a virulent virus strain; on the other hand, they may reach a stage, after several attacks, when they will exhibit solid resistance to attempts of re-infection. With the sole exception of the latter relatively small group, all horses are more or less susceptible to infection and re-infection with the anemia virus. The peculiar distribution of cases noted within a given area has often been discussed and the problem analyzed by several authors. Beside noting the seasonal occurrence of the outbreaks during the late summer months and in the autumn, search for a common regional factor was long in progress, since the disease was known to persist in certain well-defined areas in many countries. Lamarre ( 2 3 4 ) considered the geologic formation of the terrain an important factor in the epizootology of the disease. (8)

2.

N A T U R A L TRANSMISSION OF INFECTION

9

D o b b e r s t e i n and H e m m e r t - H a l s w i c k ( 8 5 , 8 6 ) concluded, after an extensive search into the p r o b l e m and a detailed discussion, that neither the g e o logical structure, n o r the watercourses of a g i v e n region appeared to h a v e outstanding significance in influencing the dissemination of the infection and the incidence of the disease.

In A m e r i c a outbreaks occurred chiefly

in undrained, l o w - l y i n g , s w a m p y

areas

( h e n c e the name s w a m p

fever)

such as, f o r e x a m p l e , along the valley of the R e d R i v e r in M i n n e s o t a and N o r t h D a k o t a , in the valleys of W y o m i n g , in the Mississippi Delta, and along the wet coastal plains of the Gulf of M e x i c o in T e x a s ; outbreaks m a y occur, h o w e v e r , in any locality regardless of altitude and drainage, v i d e d that infected animals are present or brought into such and circumstances (425).

are f a v o r a b l e

f o r transmission

susceptible

horses

T h e influence of g e o l o g y and the seasonal incidence in

France

w e r e recently carefully analyzed by L u c a s

(262).

to

pro-

territories

Steck

(419)

believes

that the local climate has some bearing o n the regional distribution

and

the occurrence of outbreaks. The

first

record

about the successful experimental

transmission

of

infectious anemia is in the report, f o r 1902, of the M i n i s t r y of A g r i c u l t u r e in C a n a d a

( 4 5 7 ) ; according to this report, b l o o d f r o m t w o horses sick

with the disease ( s w a m p f e v e r ) was inoculated into a healthy horse that reacted with a rise of

temperature o n the

subsequent rises 10 to 12 days apart. were

similarly

12th day and exhibited

In E u r o p e , V a l l é e and C a r r é

successful in reproducing

the disease

by

two

(467)

inoculation

infectious b l o o d , and also by bacterium f r e e filtrates of the b l o o d

of

serum.

W h i l e these experiments established the infectious nature of the malady, they

did not c l a r i f y the question concerning

disease spreads under natural conditions.

the manner

in which

the

Since observations o n naturally

infected horses merely suggest possible w a y s of infection, experiments w e r e d e v i s e d by a n u m b e r of authors in o r d e r to p r o d u c e experimental e v i d e n c e f o r o r against the various concepts advanced with regard to the m o d e of natural

transmission.

Infection C a r r é and V a l l é e

by Way of the Alimentary

Canal

( 5 6 , 5 8 ) , analyzing the possibilities of the spread

of the disease as it occurred in France, c a m e to the conclusion that sanitary conditions in the stables are mainly responsible, and that the alimentary tract might serve as the portal of entry f o r the virus, b y means of drinking water and f o o d of the animals. the

disease

in normal

horses

by

feeding

amounts of urine, or diarrhoeic

(bloody)

infected

was

findings

animals.

Their

concept

They them

were

able to

blood,

large

f e c a l material collected

from

accepted

infectious

the

reproduce

and, in the

w e r e soon c o n f i r m e d in E u r o p e ( 3 8 8 , 1 6 0 ) , A m e r i c a

main,

their

(124,470),

10

EQUINE INFECTIOUS

ANEMIA

and Asia ( 1 9 1 ) . Nevertheless, attempts by others to transmit the disease by feeding feces from infected horses met with no success ( 4 7 0 , 1 9 1 ) . Van Es, Harris and Schalk ( 4 7 0 ) transmitted the infection by intravenous and by subcutaneous injection of blood, of urine, and of filtered serum coming from infected horses, and also by giving the urine per os, and by feeding the horses capsules containing virulent blood. However, they were unable to incite the disease in normal horses by giving them subcutaneously 100 cc. of an extract of feces from infected animals, or larger amounts of the same by a stomach tube. Manninger ( 2 7 3 ) infected horses by feeding them large amounts of blood, or urine. The infectiousness of urine given per os was found to be inconsistent or weak (191,273) and was related to albuminuria ( 5 8 , 2 2 0 ) . All attempts at oral transmission with urine by the U. S. Bureau of Animal Industry failed, as reported by Stein and co-workers ( 4 3 8 ) . It seems doubtful whether the large oral doses of urine (up to 2500 cc.) used for experimental transmission are frequently duplicated under natural conditions, even in crowded stables kept in very poor sanitary condition; and it is extremely unlikely among horses at pasture. The fact that the disease does not spread readily by contact casts doubt upon the assumption that the alimentary canal is the main route of entry for the virus. Chances of the contact infection were checked by Francis and Marsteller (123,124) who kept a pony continually exposed to infection for more than two years by intimate association with infected horses; no sign of infection was noted in the pony and its blood was non-infectious when tested by horse inoculation. A number of general observations are on record in the literature with a similar conclusion (160, 444,127,264,399,222,438). Instances are cited that normal horses that were in daily contact with infected animals, or were quartered in stalls adjacent to those occupied by infected horses, did not contract the disease in spite of long exposure, especially if the animals were kept in clean stables, free of bloodsucking insects. This suggests that there must be modes of transmission other, and possibly more important, than by way of the alimentary tract. The Role oj Vectors in

Transmission

There is no doubt that infectious anemia can be experimentally transmitted by minute amounts of virulent blood, which is in keeping with the behavior of blood-borne virus diseases. A logical inference would be then the possibility of natural transmission by the intermediate action of bloodsucking insects. Indeed, the seasonal incidence of the disease strongly points to the possible role of some insect vector most abundant in stables and on pastures during the summer and autumn months. Brimhall, Wesbrook and Bracken ( 4 6 ) in America suggested, previous to the publication of Vallée and Carré's work, the role of insects, when

2.

N A T U R A L TRANSMISSION OF I N F E C T I O N

11

considering the mode of natural transmission from the standpoint of pasture conditions in Minnesota and the seasonal incidence of the disease; they took account of reports that fresh outbreaks of the disease occurred only in the summer or early autumn, and were more frequent during rainy summers. Mack ( 2 7 1 ) also considered the role of biting flies and mosquitoes as a possibility, and Mohler ( 3 0 2 ) regarded transmission of the disease by insects as a definite likelihood, according to the report of the Bureau of Animal Industry for 1908. Francis and Marsteller ( 1 2 4 ) attempted unsuccessfully to transmit the infection through ticks (Boophilus annulatus). The observations of the Japanese Commission ( 1 9 1 ) , while investigating the problem of transmission, did not establish the role of a particular species; nevertheless, they seemed to implicate insects, and their conclusion was that, in Japan, horseflies are "the real transmitters." The first well-controlled experimental transmission by insects was published in 1914 by Scott ( 3 9 5 ) , who demonstrated at the Agricultural Experimental Station in Wyoming on horses kept in screened cages that the stable fly ( S t o m o x y s calcitrans) can readily transmit the virus from infected to normal horses. Later he reported, at a meeting in 1917, experimental proof that the infection may also be transmitted by horseflies (Tabanidae). Howard ( 1 7 5 ) published experiments in the same year, the results of which suggested that infection has been transmitted from one horse to another by stable flies, while his experiments with horseflies gave negative result. The possible role of both, stomoxys and tabanus flies, has been experimentally confirmed, among others, by Liihrs ( 2 6 4 ) and Fortner ( 1 1 6 ) in Germany, by Lehnert ( 2 4 5 ) in Sweden, by Zenkner and co-workers ( 4 9 5 ) in Poland, and by Laktionov and co-workers ( 2 3 1 ) in Russia. Liihrs successfully transmitted the infection by using the stomach and intestinal contents of stable flies, and of some tabanids as well, which were collected in stables of infected horses; however, results were negative when he let such stable flies bite susceptible horses. Fortner achieved transmission of the infection by allowing infected tabanid flies to bite the horses repeatedly, while Zenkner transmitted the disease by use of the mouth parts of stomoxys flies that were collected while feeding on an infected horse. Lehnert ascertained the role of insects in transmission by showing that horses in well-screened stables did not contract the disease. Liihrs ( 2 6 4 ) reported significant results noted in experiments with mosquitoes; positive results were recorded by injecting normal horses with stomach contents of mosquitoes ( A n o p h e l e s maculipennis) collected in stables of horses suffering from infectious anemia, and also by allowing the mosquitoes to bite a susceptible horse; the larvae of mosquitoes did not contain virus. Scott ( 3 9 8 ) tried to confirm these findings in America, but his attempts of transmission by Aecles mosquitoes were unsuccessful. Panisset ( 3 4 0 ) reviewed in 1922 the evidence related to insect transmission.

12

EQUINE INFECTIOUS

ANEMIA

Most of the above findings have been later confirmed a n d extended by the U. S. Bureau of Animal Industry. Stein and co-workers ( 4 2 8 , 4 2 9 ) have shown in a series of carefully planned experiments that t h e horseflies (Tabanidae) and the stable fly ( S t o m o x y s calcitrans) can communicate the infection to normal horses. They also demonstrated that mosquitoes (Psorophora columbiae) can transmit infectious anemia in an i n a p p a r e n t , subclinical form. A similar subclinical infection was p r o d u c e d in a susceptible horse by the injection with 1 cc. of a 1 : 1 0 0 , 0 0 0 dilution of infectious serum. Their papers contain a good review of the literature dealing with the problem of insect transmission. Leeches have also been considered as potential transmitters in lowlying, swampy regions. Indeed, Miegeville ( 2 8 9 ) collected leeches ( L i m natis nilotica) from the legs of anemic horses and reproduced the disease in a susceptible animal by the use of the "press-fluid" of the insects. Glattli ( 1 4 0 ) regarded leeches as possible vectors in spreading t h e infection. Another ectoparasite of the horse similarly b l a m e d is t h e louse, Trichodectes equi (Bovicola equi) ( 4 0 1 ) . T h e role of the insects in the transmission is apparently a mechanical one ( 3 9 6 ) . It seems that any type of insect that first feeds u p o n a diseased horse and then upon a normal animal is a potential carrier and transmitter of the virus. It is immaterial whether the insect belongs to the ectoparasites or the endoparasites. It has been shown experimentally that the parasitic worm, Filaria equina (Setaria equina), and its eggs a n d e m b r y o s contained virulent, infectious virus ( 2 6 4 ) , and so did adult strongyles ( 4 3 0 ) ; these have been collected from horses affected with infectious anemia. O n t h e other hand, extract of third-stage larvae of strongyles originating f r o m the feces of infected horses, and similarly an extract of washed larvae of the so-called botfly (Gaslerophilus intestinalis), collected f r o m the s t o m a c h of a horse that died of acute infectious anemia, failed to p r o d u c e s y m p t o m s when injected into normal horses ( 4 3 0 ) . Products of insects and their larvae have been thought to be directly responsible for infectious anemia. Ries ( 3 7 2 ) first suggested this possibility, and Seyderhelm and Seyderhelm ( 4 0 3 , 4 0 4 ) later presented experimental studies which they believed would indicate that a toxic p r o d u c t ( O e s t r i n ) of the larvae of botflies (Gastrophilus haemorrhoidalis and G. equi)* can produce genuine infectious anemia. Extracts of such larvae, collected from the stomach of slaughtered horses, provoked fever reaction and other symptoms including anemia when injected into n o r m a l horses. No experimental proof was presented, however, that they excluded t h e presence of virus carriers among the horses f r o m which the larvae were collected. In some of their experiments, heated extracts also p r o d u c e d suggestive symptoms. * R e c e n t l y a d o p t e d scientific t e r m s :

pliilns intestinalis.

Giisteropliihix litiemorrhoidalis

and

Gastero-

2.

NATURAL TRANSMISSION OF INFECTION

13

The conclusion of the Seyderhelms was soon rejected by Carré and Vallée ( 5 9 , 6 0 ) . Theiler reported in 1916 that he was unable to confirm their findings. Similar was the experience of other re-investigators, like Van Es and Schalk ( 4 7 1 , 4 7 2 ) , Klempin ( 2 0 6 ) , de Kock ("209), and du Toit ( 4 5 2 ) . They reproduced infectious anemia by use of extracts of larvae from infectious anemia cases, but they failed to reproduce the typical disease with extracts of bots from normal horses. Van Es and Schalk's work indicated that parasitic allergy can produce transient symptoms somewhat resembling those of equine infectious anemia ( 4 7 2 ) . R. Seyderhelm ( 4 0 6 ) emphasized in 1917 that the Oestrin "as a genuine blood poison" is responsible for pernicious anemia cases of parasitic origin only. Marxer ( 2 8 2 ) believed that the toxic effect of gasterophilus larvae is anaphylactic in nature as shown by experiments in rabbits.

Various

Other

Routes

of

Transmission

The possibility of many other routes and means of transmission has been explored by several investigators experimentally. The disease was successfully transmitted by nasal secretions ( 3 9 9 , 5 4 6 ) , by application of the virus to wounds and to the conjunctiva ( 3 5 9 ) , and by injecting infectious serum into the anterior chamber of the eye ( 3 1 9 ) . Manninger ( 2 7 3 ) infected horses by the application on their legs of bandages soaked with infectious blood, or urine; on the skin where the bandages were applied, there were no wounds or erosions detectable by the naked eye. Beller and Traub ( 5 4 6 ) were unable to demonstrate transmission through the intact skin. Scott ( 3 9 6 ) demonstrated in a simple model experiment, how easily accidental infection can occur through abrasions of the skin or through surgical manipulations. He reproduced the disease in a susceptible horse with a single prick by a hypodermic needle that was contaminated through first pricking an infected animal. It is apparent from this, that careless use of unsterilized surgical instruments, syringes, and hypodermic needles may well play a role in disseminating the infection among horses. Infection through the skin by the intermediary of harness has been described; and the use of common equipment such as bridles, currycombs, and brushes has also been incriminated in the spread of the disease ( 4 3 8 ) . Many of the accidental infections can be readily explained in face of Scott's experiment just mentioned, or if we recall the experiments with extremely diluted infectious serum ( 4 2 9 ) . As mentioned above, nasal secretions may contain the virus. Thus, it is conceivable that infected horses or virus carriers can transmit the disease through the respiratory system under suitable conditions, such as at times of catarrhal symptoms in the upper respiratory tract whether the

14

EQUINE INFECTIOUS

ANEMIA

catarrh is caused by the virus of infectious anemia or by other infectious agents. T h a t the nasal mucous membrane may be attacked by the virus is suggested by the observation that two of our experimental horses suffered from severe capillary nose bleeding lasting for over a week during febrile attacks. It is felt, however, that involvement of the nasal mucosa or its capillary system is not a regular feature in infectious anemia; but it may develop in consequence of the petechial hemorrhages observed on different mucous membranes in toxic cases. Congenital

Transmission

of the Infection

to Foals

T h e transmission of the virus by infected mares to their offspring is a complex problem. Observations have been reported suggesting the passage of the virus to the fetus through the placenta ( 4 3 1 , 4 4 0 , 4 6 0 ) . T h e milk of infected mares contains the virus as shown by parenteral injection to susceptible horses ( 1 9 1 , 3 9 9 , 4 3 1 ) , but whether the virus ingested by the milk remains viable in the stomach before it comes to resorption is another question. No conclusive evidence was furnished by investigations of Stein and Mott ( 4 3 1 , 4 3 3 ) as to whether some foals acquired the infection from being nursed by their infected dams, as part of their experiments suggested, or by close contact with their dams. T h e semen of infected stallions may contain the virus; subcutaneous inoculation of such semen into a normal mare resulted in a subacute form of infection ( 4 3 1 ) . T r a u t wein and Schmidt ( 4 6 0 ) observed a severe outbreak among foals, ten days to nine weeks of age, born of infected mares. T h e y stated that colts are more susceptible to the virus than adult horses. Krauss ( 2 2 3 ) described an epizootic believed to have originated from five imported stallions that were virus carriers. The problem of congenital transmission of the infection is probably closely connected with the fact that immune bodies and other blood proteins of the pregnant mare are, as a rule, not transferred to the fetus during the intrauterine life ( 3 8 9 ) , at least not to the same extent as in some other animal species. However, by means of the colostrum, such proteins are readily available to the foal ( 3 8 9 ) ; the same was shown by experimental work performed by Wallenstein, Brueckner and Poelma ( 4 7 9 ) in connection with the Rh-like isoagglutinins of newborn foals. If similar conditions prevail in infectious anemia, as we believe they do, foals may be infected, probably subclinically, during the intrauterine period, or during the first days of their life after birth, when colostrum may pass undigested (and the virus contained in it unaffected) through the mucous membranes of their intestinal canal. It is known that, at this developmental stage, the permeability of these membranes is different from that during thè rest of the life.

2.

N A T U R A L T R A N S M I S S I O N OF I N F E C T I O N

15

Oppermann ( 3 2 6 ) called attention to the fact that infectious anemia is responsible for many abortions in diseased mares. Stein and Mott ( 4 3 3 ) , on the other hand, stated that mares, when experimentally exposed to infection while pregnant, may or may not abort their fetuses during fever attacks. *

It is now generally accepted that infectious anemia spreads mainly through the agency of insects, in particular of bloodsucking flies ( 1 4 7 , 2 0 1 ) . This conclusion has been reached through experimental research and by logical inference from the seasonal appearance of fresh outbreaks, coincident with the seasonal activity of biting flies and mosquitoes ( 3 9 7 ) . It may be added that there are several virus diseases known to be transmitted by bloodsucking insects; for example, yellow fever is spread in tropical regions through mosquitoes, and Kelser ( 1 9 6 , 1 9 8 ) demonstrated experimentally that mosquitoes ( A e d e s aegypti) can transmit equine encephalomyelitis, a neurotropic virus disease of horses, that has been shown to be transmissible also to man (Meyer ( 2 8 7 ) ) . The source of virus in the spread of infectious anemia is the sick horse or the virus carrier. It is known that once a horse is infected, it remains a life-long carrier of the virus. Several authors tested the blood of such carriers systematically for 12 to 16 years, and noted positive results ( 1 1 6 , 1 1 9 , 2 2 1 , 3 8 0 , 4 6 2 ) . An unsuspected virus carrier among horses, naturally, constitutes a dangerous reservoir of the virus and continues to spread the disease until eliminated from the group. There is no indication that an intermediate reservoir of the virus exists. The role of insects is considered plainly as a mechanical transfer of the infectious material, usually blood, and no evolutionary cycle is postulated in the development of the virus within the insects. Whether some insects or parasitic worms may harbor the virus in a masked form for a long time is not known. Shope ( 4 1 0 ) , in a series of interesting experiments, has demonstrated that swine influenza virus can survive in lungworm larvae of the swine, or in earthworms in masked form for at least two years. The possibility of such latent, masked persistence of infectious anemia virus in an intermediate host has not been explored so far; it could explain the repeated outbreaks on pastures where the disease notoriously flares up every year, and yet the infection cannot be traced to virus carriers. Although the most important, insect transmission is not the only mode responsible for the spread of the disease. Many authors believe that the disease has little or no tendency to spread among horses by contact under good hygienic conditions. On the other hand, infection can readily occur through abrasions of the skin, or via the mucous membranes of the animal, and by accidents capable of bringing the virus to resorption.

16

EQUINE INFECTIOUS ANEMIA

If the infectious material does not reach the blood stream quickly, either directly, or by resorption, it is probably retained in the regional lymph glands and is apt to produce a mild, latent type of infection, or to bring about the usual clinical symptoms after a prolonged incubation period. It appears, as if merely a sensitization had been achieved first, with no manifest symptoms, to be followed by clinical symptoms later on. Steck (420,421) states that, in certain localities where infectious anemia is enzootic, many latent infections occur among horses that have frequent contact with one another. Such cases are usually not recognized until some provocating factor activates the disease and brings about manifest symptoms in the horse. According to the above concept, the latent, subclinical infection may exist for a long time before it is recognized on the basis of clinical symptoms, and then it is regarded as a relatively "rare" instance of contact infection. It may be said in conclusion then that natural transmission of infectious anemia can take place in many different ways, the most common of which seems to be insect transmission; but there is sufficient evidence that the disease may spread by contact and by a number of other ways of accidental infection.

CHAPTER 3

SYMPTOMATOLOGY As a study of the symptoms of infectious anemia was not one of our prime objectives, we shall report only a few pertinent observations made on horses that had been injected with material known to contain virulent virus. The acute cases comprise those animals suffering severe symptoms that either terminated fatally or the horses were destroyed in the early phase of the disease. In this stage the febrile attacks either occurred at short intervals (one to four days) or the fever continued unabated until death. The incubation period varied from 8 to 17 days. The peak of the temperature at the time of the febrile reaction ranged from 104° to 108° F. (40° to 42.2° C . ) . The pulse was 46 to 56 per minute when taken on the resting animal. Symptoms usually accompanying the exacerbations were thirst, often perspiration, debility, and depression. Epistaxis, sublingual and nasal hemorrhages, were occasionally observed. Pallor of the mucous membranes was noted only when the anemia was severe. A papular eruption was observed on the neck of one horse at the time of the second attack; the rash, which lasted four days, spread over the entire body. In fatal cases a marked debility was indicated by the animal's inability to rise from a lying position. The acute phase of the disease was usually less than a month in duration; it either terminated in death or, evidencing gradually less severe attacks, changed its character to become a subacute or chronic case. Subacute cases consisted of those horses repeatedly manifesting fever paroxysms at afebrile intervals of 6 to 24 days for a longer period of time, usually for several months. The attacks were milder; the temperature ranged from 102° to 105° F. (38.9° to 40.6° C . ) * during attacks and the accompanying symptoms were similar to those observed during the acute phase. Many of our horses were remarkably free of severe symptoms during the first and second febrile attacks; symptoms became more notice4 For converting temperature readings given in Fahrenheit degrees into centigrades ( C ° ) , see Table 1.

(17)

18

EQUINE INFECTIOUS

ANEMIA

able and increased in severity during subsequent attacks in spite of the usually milder fever response. Loss of weight, depression, marked debility, edema of the dependent parts, and unsteady gait were most apparent after the second month. In this stage, pallor of the mucous membranes was usually quite apparent. TABLE 1 C O M P A R A T I V E T E M P E R A T U R E F I G U R E S IN D I F F E R E N T S C A L E S

Fahrenheit (F°)

Centigrade (C°)

(F°)

Centigrade (C°) 39.5

Fahrenheit

97.0

36.1

103.0

97.5

36.4

103.5

39.7

98.0

36.7

104.0

40.0

98.5

36.9

104.5

40.3

99.0

37.2

105.0

40.6

99.5

37.5

105.5

40.8

100.0

37.8

106.0

41.1

100.5

38.0

106.5

41.4

101.0

38.3

107.0

41.7

101.5

38.6

107.5

42.0

102.0

38.9

108.0

42.2

102.5

39.2

108.5

42.5

In the chronic form, the febrile periods occurred at intervals of one month or more and were less severe. Chronic cases may be divided into two groups: those animals whose condition gradually grew worse, and those maintaining a relatively favorable state of health. In the former group, emaciation, edema of dependent parts, incoordination, depression, and debility were prominent characteristics. In the latter, the animals appeared healthy and produced no manifest symptoms, but their temperatures, taken twice daily, revealed mild elevations at about monthly or longer intervals. Mortality.—Accurate figures on mortality are lacking because animals afflicted with inapparent or mild forms of the malady would escape diagnosis; consequently no dependable figures are available to serve as a basis for calculation. Nevertheless, some estimates have been made in various countries and a few of the figures reported in such estimates should stand here. Watson ( 4 8 0 ) emphasized the high mortality, when reporting in 1896 about the disease as seen in Wisconsin. Francis and Marsteller ( 1 2 3 ) stated in 1908 that the mortality was 80 per cent in Texas. The U. S. Bureau of Animal Industry ( 4 6 2 ) , reporting on the outbreak of infectious

3.

SYMPTOMATOLOGY

19

anemia in a group of several hundreds of valuable thoroughbred horses during 1947, made the statement that "this outbreak in New England took a toll of 80 horses." In the Province of Quebec, Canada, the mortality was set at 31 per cent in 1941, and 21 per cent in 1942. In Switzerland, Wyssmann, in 1915, estimated the mortality 57 per cent from a series of cases studied. In the enzootic areas of France, the mortality was considerable ( 1 8 1 ) . In 1933, 30 horses died of infectious anemia at one single establishment of the army in Belfort, France. In Württemberg, Germany, 65 animals died of the disease in 1931, and 218 in 1932. The Japanese Commission ( 1 9 1 ) reported a mortality of 30 to 70 per cent depending upon the form of the disease, the climate, and the care of the animals. They stated that, during a summer month in 1911, of 107 horses present in one village a total of 62 were lost, of which 24 died, and 38 were destroyed. *

The symptoms exhibited by our experimental horses were essentially the same, although not as severe as those reported by other investigators. The reason for this difference in severity is not quite clear but it may partly be attributed to the feed and care that our animals received. While under experiment, our horses were housed in screened isolation stables and were not subjected to work or any physical strain. Division of the disease into acute, subacute, and chronic forms was made by Carré and Vallée ( 5 8 ) on the basis of the fever attacks. In the acute stage, there were usually no remissions, the attacks were severe, and the duration of the phase was from five to fifteen days. In the subacute phase the symptoms were attenuated, remissions were clear-cut and the duration was a few weeks to several months; whereas in the chronic form, fever attacks were infrequent and the symptoms were those of extreme progressive anemia. Hutyra, Marek and Manninger ( 181 ) characterized the three divisions on the same basis but in greater detail. They reported the duration of the acute phase in adult animals as three to four weeks with no remissions or only a few at very short intervals; in the subacute phase, attacks occurred two or three times a month and the duration of the stage was a few weeks to several months. Febrile attacks in the chronic form were milder and recurred at intervals of one to three months or more over periods lasting from months to years. These authors emphasized that one animal may pass through all three stages of the disease, moreover that the disease, after becoming quiescent, may resume an acute, fulminating course. Other classifications found in the literature do not vary essentially from the above (28,191,264,426,470).

20

EQUINE INFECTIOUS ANEMIA

There are reports that the incubation period as observed in artificially induced infections may vary from one day to three months or more (28,181,191,426,470) depending upon what is taken as the first sign of infection. The most frequently observed incubation usually extends from one to three weeks (181,426,470). Martin ( 2 7 8 ) concluded from his observations on experimentally inoculated animals that the actual period of incubation is two to five days, at which time the first reaction is observed in the form of a mild temperature rise; the first marked fever reaction occurred in ten to nineteen days. Gerlach ( 1 3 2 ) noted incubation periods lasting as long as three months. His experimental animals were hyperimmune serum horses; this may account for his findings, as such animals have high immune globulin level. On the basis of our experiments, we believe that the fever reaction to the virus is delayed in such cases because of the high unspecific immune globulin, as discussed later in the chapter dealing with the pathogenesis. Factors which are believed to influence the length of the period between inoculation and the appearance of symptoms are the serial passage of the virus ( 5 8 , 2 1 7 ) , the route of injection (271,398,470), and the material containing the virus, i.e., whole blood, serum, urine, milk (191,219,398, 4 7 0 ) . Variations in the virulence of the virus and differences in the susceptibility of the experimental horse are factors as yet not sufficiently understood ( 2 8 , 4 7 0 ) . According to Stein, Lotze and Mott the incubation period was lengthened by dilution of the virus as the dilutions became greater. A latent form of the disease was produced by inoculation of a 1:100,000 dilution or through transmission by mosquitoes ( 4 2 9 ) . These authors also provided evidence that the incubation period was lengthened when transmission was through stable flies ( 4 2 8 ) . With the exception of the rapidly fatal acute and the latent chronic phases, the disease has as its main characteristics: intermittent fever attacks, progressive debility, marked depression, emaciation, and edema of dependent parts (28,181,191,470). Other symptoms reported as occurring in the course of the disease include lack of appetite (181,191,426), nocturnal polyuria (46,123,181, 4 2 6 ) , diarrhea (58,181,426), increased irritability ( 2 8 , 1 8 1 ) , incoordination ( 2 8 ) , weakness or paralysis of the hind quarters (123,181,337,426), pallor of mucous membranes (28,181,470), yellowish discoloration of the conjunctiva (28,123,271,426), urticaria ( 1 5 2 ) , sublingual and nasal petechial hemorrhages (257,417,3), abnormal cardiac activity ( 1 9 1 , 4 2 4 ) , accelerated pulse (181,191,426), increased respiratory rate ( 1 9 1 , 4 2 6 ) , and also splenomegaly as detected by rectal palpation ( 1 9 1 ) , just to mention a few references from the voluminous literature on symptomatology of equine infectious anemia.

3.

SYMPTOMATOLOGY

21

Symptoms in the chronic and latent cases have been studied by Zeller ( 4 9 4 ) and by several other investigators; it has been concluded that symptoms in this stage are not distinctive for the disease. However, when combined with the history of the case and hematology, they should arouse suspicion. Apparently recovered and symptomless cases have been known to undergo relapses when exposed to strenuous labor ( 1 8 1 ) , inclement weather ( 1 9 1 ) , and surgical manipulation ( 4 2 6 ) , or by profuse bloodletting ("provocation").

CHAPTER 4

CLINICAL

DIAGNOSIS

The only definite means of diagnosis of equine infectious anemia which is now available is the horse inoculation test. On account of the expense involved, this is utilized only if the sick horse is valuable or there are other reasons for which it is essential to arrive at a definite and final diagnosis in a horse suspected to suffer from infectious anemia. T h e test consists of inoculating blood from the suspect case into a normal horse, and its result is considered positive, if characteristic symptoms develop after an incubation period. In many cases, however, the diagnosis can be made with a reasonable degree of accuracy by considering collectively the history of the case, clinical symptoms and the results of blood examination, particularly in the acute and subacute stage of the disease. Diagnosis may be difficult in an early severe case or in a chronic carrier. It may be relatively easy when a new case with typical symptoms appears in an endemic area. A tentative diagnosis is often made when signs of anemia are present and fever is observed, but it hardly is necessary to point out that more data than these are requisite before arriving at this conclusion. When the disease takes a rapidly fatal course, it may closely resemble acute septicemia ( 1 8 1 ) . The first attack may persist as a high continuous fever with death occurring before the usual sequence of symptoms develops. Anemia may be absent or mild. Other symptoms that may appear are marked weakness, prostration, mental depression, and slight icterus. T h e disease in the acute form may be confused with anthrax, influenza, purpura hemorrhagica, acute equine encephalomyelitis, and other acute febrile conditions ( 1 9 1 , 3 9 9 ) . According to Hutyra, Marek and Manninger ( 1 8 1 ) it may be impossible to distinguish between anthrax and infectious anemia without bacterial examination of the blood or inoculation tests. These authors noted that equine influenza spreads more quickly among a group of horses, taking a rapid and usually more favorable course than that of infectious anemia. T h e former is characterized chiefly by catarrhal manifestations. (22)

4.

CLINICAL

DIAGNOSIS

23

Other conditions which have been confused with equine infectious anemia in its early stages are piroplasmosis, pneumonia ( 1 8 1 ) , acute helminthiasis, leukemia, and neoplasms ( 4 1 6 ) . In tropical countries piroplasmosis, trypanosomiasis, as well as other protozoon diseases present a definite problem in differential diagnosis ( 2 2 7 ) . Such conditions have been reported to co-exist with infectious anemia ( 3 5 0 ) . In piroplasmosis there is usually icterus and often hemoglobinuria. In piroplasmosis there are petechial hemorrhages on the conjunctiva, whereas in infectious anemia they are sublingual. In piroplasmosis large numbers of parasites are found in the blood at the time of the fever peaks. Hematologic examination of the blood in early stages of infectious anemia should be done during or right after the fever attacks, as the lowered values tend to return to normal during remissions in this phase of the disease. Sublingual hemorrhages have been claimed as a diagnostic criterion in distinguishing equine infectious anemia from other diseases (257,416, 4 1 7 , 4 2 0 ) . In equine infectious anemia the petechiae are minute and often occur in numbers ranging from hundreds to thousands. In septicemic diseases the hemorrhages are larger, and in petechial fever they vary in number and distribution, most often being found on the nasal mucosa. When the course of infectious anemia is less severe and of longer duration, the usual chain of symptoms is: intermittent attacks of fever, rapid loss of weight, debility, pale mucous membranes or some degree of jaundice, and dropsical swellings of the dependent parts, the anemia as well as other symptoms being usually most pronounced in the febrile attacks. In the subacute form, the disease may be mistaken for trypanosomiasis or strongyloidosis ( 1 8 1 ) . Other diseases from which the subacute form must be differentiated are nutritional and other types of anemia (53,179, 2 2 4 , 3 1 0 , 4 1 8 ) , filariasis ( 1 8 1 ) , chronic articular rheumatism ( 1 4 6 , 1 5 8 ) , and tumors ( 4 1 6 ) . Nutritional anemia almost always runs an afebrile course ( 1 8 1 ) . Chronic articular rheumatism has been described as showing signs of anemia and producing from time to time an irregular remittent type of fever ( 1 4 6 ) . Symptoms of joint pain may aid in differentiating the condition from infectious anemia; however, Habersang ( 1 4 6 ) reported lameness due to tendosynovitis, and Sokolowski ( 4 1 3 ) observed lumbago in infectious anemia. Differentiation from anemia due to neoplasms is illustrated by the following cases: Two horses suspected of having infectious anemia were brought to our attention. Both revealed malignant tumors at autopsy. Clinically they had displayed irregular temperature elevations, marked anemia, progressive debility, and mental depression. The lack of any regularity in the occurrence of the fever attacks, especially in the early stages, as well as the past history of the case are factors which might

24

EQUINE INFECTIOUS

ANEMIA

assist in making a differentiation. In the cases just mentioned, o u r laboratory tests (hemagglutination and blood protein d e t e r m i n a t i o n ) were helpful in ruling out infectious anemia. In chronic cases in which the characteristic symptoms of progressive emaciation, debility, anemia, and intermittent fever attacks f r o m time t o time are apparent, diagnosis is not difficult. However, in the symptomless, balanced virus carrier the disease easily escapes detection; during this stage, the animal may look as healthy as any other horse. T h o u g h it h a s been claimed that a constant lymphocytosis is present in such horses, this is apparently not always true, and even so is not sufficient to establish a criterion of carriership. T h e r e have been n u m e r o u s reports of mixed infections of infectious anemia and tuberculosis, such as those by E i l m a n n ( 1 0 5 ) , G h i e r ( 1 3 3 ) , L a m a r r e ( 2 3 4 ) , Panisset and co-workers ( 3 4 1 , 3 4 2 ) , and others. B e h r e n s ( 3 5 ) reported that seven out of eight horses, suspected of having infectious anemia on clinical examination, were shown at autopsy to have tuberculosis. O n e must be careful in making or rejecting the diagnosis of infectious anemia on the basis of the presence o r absence of a positive tuberculin test. Several investigators reported positive tuberculin skin tests in a large percentage of their infectious anemia cases ( 1 0 5 , 3 4 1 ) . However, in most of them the two diseases might have been co-existent in the same animal. Panisset and Pflieger ( 3 4 2 ) came to the conclusion that the tuberculin test is of no value in the differential diagnosis of infectious anemia. Stein ( 4 2 6 ) stressed the fact that, although chronic parasitism c a n present similar symptoms, it is also possible that intestinal parasitism a n d infectious anemia may be present in the same horse. A n aid in their differential diagnosis is the microscopic examination of the feces f o r eggs of intestinal parasites and examination of blood films for eosinophilia. O n e should bear in mind, however, that changes occur in the n u m b e r of eosinophils also in infectious anemia. *

Beside analyzing the aggregate of clinical symptoms, various tests can be used as additional tools of the diagnosis. T h e problem of differential diagnosis on the basis of histopathologic findings has been investigated by many workers, a m o n g them Ziegler ( 4 9 6 ) , Noller a n d D o b b e r stein ( 3 2 2 ) , O p p e r m a n n ( 3 2 8 ) , Hjiirre ( 1 6 8 ) , Moszczenski a n d K r u p s k i ( 3 1 0 ) . and Steck and H a u s e r ( 4 2 3 ) . Liver biopsy has also been advocated to decide diagnosis in t h e living animal. Richters ( 3 6 9 ) , Wall ( 4 7 8 ) , H j a r r e ( 1 6 8 ) , and m a n y others reported the results of this method for histological diagnosis. Wall ( 4 7 8 ) made suggestions for modifying the trocar to be used f o r this p u r pose. A k e r s t r o m ( 3 ) believed that liver biopsies are a valuable aid in making a diagnosis and implied that the p r o c e d u r e was relatively safe

4.

CLINICAL DIAGNOSIS

25

and easy to perform. He noted that sublingual hemorrhages and liver biopsy were of some value in detecting carriers. Histological examination of the tissues of slaughtered horses has been practiced for some time to determine the incidence of infectious anemia in a certain area, and several reports have been published on the subject. However, as many investigators rightfully pointed out, it is quite false to make a diagnosis of infectious anemia in the abattoir without the knowledge of the clinical history of cases, as many other conditions may cause similar pathologic changes, particularly hemosiderosis in the liver ( 3 7 5 ) . Tests other than histological investigation that might be helpful in supporting the clinical diagnosis will be considered in the chapters on Hematology, Hemagglutination, and other Laboratory methods of diagnosis. Taken separately the clinical, laboratory, and pathological findings can serve merely as suggestions for tentative diagnosis. Combined with one another, however, they often can be of definite value in deciding the differential diagnosis. Nevertheless, the horse inoculation test remains the only means of assuring, beyond any doubt, a final diagnosis of infectious anemia.

CHAPTER

5

PROPERTIES OF THE

VIRUS

While some of the main characteristics of the virus of infectious anemia are known, many of its properties have not been sufficiently explored as yet. T h e reason for this is the lack of simple and inexpensive methods of demonstrating the presence of the virus in a material u n d e r study. T h e horse inoculation test is the one dependable way of indicating the presence of an active virus strong enough to provoke clearly recognizable symptoms; but, for obvious reasons, the test cannot be utilized to any great extent. In order to determine the ultimate strength of a virus, inoculation of graded dilutions into a considerable n u m b e r of experimental animals is required. Such titration is, in most cases, not feasible in infectious anemia; neither is the application of indirect methods, which are readily available for the study of many other virus diseases. A brief s u m m a r y will be given below on what is k n o w n at present with regard to filterability, size, and biological identification of the virus; its distribution within the body fluids and tissues; its resistance to physical and chemical agents; and finally, current views concerning its nature. Filterability

and Size

The virus etiology of the disease has been established by the experiments of Vallee and C a r r é ( 4 6 8 ) . They transmitted the infection to n o r m a l horses with infectious blood and were equally successful in reproducing the disease by giving a susceptible horse 100 cc. of a bacterium free filtrate of horse serum obtained f r o m a case of infectious anemia. T h e y determined the efficiency of the filter in a collateral experiment; it removed all o r g a n isms from a highly infectious broth culture of Pasteurella, one of the smallest among pathogenic bacteria, as verified by a test in guinea-pigs. T h e filterability was soon confirmed by several authors, a m o n g them Ostertag ( 3 3 8 ) , Hempel ( 1 6 0 ) , and Theiler and Kehoe ( 4 4 6 ) . Balozet ( 2 7 ) determined the approximate size of the virus by h o r s e inoculation experiments following filtration through Elford's collodion m e m branes of known porosity. He estimated the size of the virus to be between 18 and 50 m/t, since it passed through m e m b r a n e s having a p o r e size of (26)

5.

27

PROPERTIES OF THE VIRUS

100 iTi/i, but was retained by those of 52 m^. Reagan and collaborators ( 3 6 4 ) attempted to determine the size and shape of the virus by means of the electron microscope; in the sediment of an infectious horse serum filtered and then centrifuged at 52,000 rpm for 3 hours, they noted spheric formations ranging from 11 to 59 nip (average 30 m,u), which they considered the virus of infectious anemia, since no particles of similar size were noted in normal horse serum. The size thus determined seems to be in fairly close agreement with that found by Balozet. On account of its very small particle size, the virus cannot be detected through the use of the ordinary light-microscope, as the diameter of the virus remains much below the resolving power of such microscopes. Inclusion bodies have not been demonstrated in infectious anemia. The small, ringlike formations to which first Mack called attention were regarded as artefacts by Swingle ( 4 4 4 ) . No bacterial organism was ever cultivated with any regularity, or found to be present in the blood or various organs in horses suffering from infectious anemia. Miyagawa and co-workers ( 2 9 2 ) reported the cultivation of spirochetes on Noguchi's medium from the blood of infectious anemia horses, but Hoskins ( 1 7 4 ) , Mocsy ( 2 9 4 ) , and others were not able to confirm their findings. If we consider that Atoxyl and various Salvarsan preparations have been found ineffective in the therapy of the disease, this fact itself would indicate that infectious anemia is not caused by spirochetes or protozoa. Such infections, however, may very well co-exist with infectious anemia, or may be mistaken for it, as Abbondanza ( 1 ) suggested. Such may be the explanation of the unusual findings reported by Pigoury and collaborators ( 3 5 0 ) , who studied equine surra (trypanosomiasis) and infectious anemia in laboratory animals. The close similarity, particularly in the clinical manifestations, between streptococcus anemia and virus anemia as observed on horses in Switzerland was discussed by Krupski, Grumbach and Leemann ( 2 2 6 ) . The question of alleged etiologic significance of parasitic toxins was mentioned in Chapter 2. Identification

of the Virus and Its Distribution

in the

Body

The virus can be identified by its pathogenic effect in susceptible animals. The typical symptoms and the differentiation of the disease from clinically similar conditions have been briefly reviewed in the preceding chapters. Additional means of studying the pathologic condition in horses experimentally infected with the virus or in animals naturally ill with the disease will be discussed in subsequent chapters. Attempts at neutralization of the virus by immune serum did not lead to any appreciable success ( 4 2 7 ) . Although differences in virulence have been noted by several authors, it is uncertain whether this is a permanent property of any virus strain. As a rule, it is influenced by the rapidity and

28

EQUINE INFECTIOUS

ANEMIA

the type of passages which it undergoes in horses ( 5 6 ) , but it is immaterial whether 1 cc. or 7 5 0 cc. of blood is injected. Strain differences of serologic type have been suspected by some research workers ( 2 9 0 , 5 4 6 ) , but it is difficult to p r o d u c e evidence for this because of the lack of p r o p e r serologic methods. T h e findings on which the assumption of strain differences is based can, in our opinion, be explained in several different ways. F o r identifying the virus, susceptible individual animals are n e e d e d ; equines (horses, mules, d o n k e y s ) are regarded as equally susceptible, although a strain adapted to one of these species within the equine family is usually more virulent to that particular species. Susceptibility of o t h e r animals than equines will be discussed in connection with the transmission experiments on laboratory animals (in C h a p t e r 1 0 ) . Although there are a few reports ( 2 6 5 , 3 4 7 , 2 2 1 ) suggesting that the infection has been accidentally transmitted to man, it is believed that such infection in h u m a n s is exceptionally rare, or else it runs an unrecognized subclinical form. Otherwise more cases would occur a m o n g persons h a v ing frequent contact with horses ill with the disease. W e may quote here an observation in our laboratory. During a post mortem examination of an experimental horse killed in the subacute stage of the disease, a laboratory worker cut his finger while cleaning a sharp surgical knife stained with blood of the horse. T h e person in question did not develop any s y m p t o m and has remained in perfect health ever since. Stein and M o t t ( 4 3 4 ) noted negative results when they tested in horses the blood of two h u m a n patients suffering from aplastic and hypoplastic anemia, which conditions have been suggested by Peters ( 3 4 7 ) to be possibly related to equine infectious anemia. Because of its constant persistence in the blood, the virus may b e regarded as ubiquiter in the body of the infected animal. Its presence has been demonstrated in the milk ( 1 9 1 , 2 6 4 , 3 9 9 , 4 3 1 , 4 3 3 ) and in nasal secretions ( 3 9 9 , 5 4 6 ) ; it is present in urine exhibiting albuminuria, as is known from the work of Vallee and Carre. Experiments would tend to show that it is not contained in saliva ( 3 3 8 , 1 6 0 , 2 5 3 ) , or in tear and sweat ( 2 8 , 2 5 3 , 3 9 9 ) . Evidence is conflicting about the virus content of feces; most workers believe, however, that it is not eliminated through the bowels ( 4 7 0 ) . N a k a m u r a and co-workers ( 3 1 9 ) tried to estimate the relative concentration of the virus in various organs of infected horses. Specific affinity of the virus to certain tissues or to a particular organ system h a s been assumed, to erythrocytes and the hematopoietic system, although it h a s not been demonstrated; but it was repeatedly stated as an inference f r o m histologic findings that the virus has a tendency to attack the capillary walls and shows a definite affinity to the r e t i c u l o e n d o t h e l i a l system. H o l z ( 1 7 1 ) , who noted in some infectious anemia cases histologic alterations of the central nervous system reminiscent of those seen in acute e n c e p h a l o myelitis has assigned a neurotropic tendency to the virus. In o n e of o u r experimental horses, which we injected with virulent virus intracerebrally,

5.

PROPERTIES OF THE VIRUS

29

no recognizable disturbance was noted in the nervous system; the horse reacted with a typical fever response and other symptoms after an incubation of 16 days. Resistance

of the Virus to Physical and Chemical

Agents

In general, the virus of infectious anemia is relatively resistant as compared with other viruses and may survive outside of the animal's body for a comparatively long time. Early reports indicated ( 4 7 0 ) that the virus is well preserved when exposed to freezing. Later work confirmed that the virus remains viable in serum or plasma for at least 6 months, if preserved at or below freezing temperature. Under bacterially sterile conditions, infectious serum may remain virulent for several months at room temperature. In our laboratory, infectious serum and plasma samples were kept frozen in rubber stoppered vials at - 1 5 ° to - 2 0 ° C . , and also at about - 7 0 ° C . on carbon dioxide-ice, without the slightest reduction of virulence in 4 years. T h e virus is fairly resistant to desiccation. According to A v r a menko ( 5 2 0 ) it remained viable on hay as long as 180 days. Investigators at the U . S. Bureau of Animal Industry found the virus fully active after 6 years storage in lyophilized form ( 4 6 2 ) . Balozet ( 2 6 ) tested the effect of ultraviolet light by irradiating virus samples in Petri-dishes with a mercury vapor lamp for 5 minutes to 7 hours; all irradiated samples remained viable as tested in donkeys. It has been known from early experiments that heating at 58° to 6 0 ° C . for one hour renders the virus noninfectious ( 5 8 , 1 9 1 ) . Inactivation of the virus for the purpose of disinfection or its attenuation for immunization was studied by many investigators. The effect of phenol ( 1 9 1 , 2 6 7 , 1 2 8 , 2 0 ) , formalin (246,475,299,300), and a number of other chemical substances (199,222,16,17,279,439) has been investigated and the products so treated were tested in horses for infectiousness. Phenol seems to possess less inactivating capacity unless it is used in somewhat higher concentration, while formalin inactivates the virus in a relatively short time. Balozet ( 2 0 ) reported that 0.1 per cent phenol inactivated the virus in 4 months, or at room temperature in 16 days ( 2 2 ) . By using 0.5 per cent phenol, serum samples kept at refrigerator temperature lost their virulence in 3 months ( 2 2 2 ) . Mocsy ( 2 9 9 ) found that 0.3 to 0.5 per cent formalin destroyed the virulence of the virus in 38 days. Glycerine first attenuated the virus, in about 4 months; while 7 months contact with glycerine inactivated it ( 1 7 , 2 0 ) . Saponin ( 1 6 ) and bile ( 1 8 , 1 9 ) were very effective in inactivating or attenuating the virus. It is thought to be alcohol resistant ( 4 5 9 ) , and according to Richters ( 3 7 1 ) strongly alkaline disinfectants are favored by Waldmann. T h e problem was reviewed and a series of experimental studies related to the resistance of the virus were reported in 1944 by Stein and co-workers ( 4 3 9 ) . In serum samples kept at 5 ° C . the virus was inactivated in a

30

EQUINE INFECTIOUS

ANEMIA

m o n t h by the addition of 0 . 5 p e r c e n t p h e n o l , o r 0 . 1 to 0 . 2 p e r c e n t f o r m a l i n , a n d also by a c o m b i n a t i o n of crystal violet a n d p h e n o l . It is o f t e n difficult to decide o n the basis of t h e test i n o c u l a t i o n in h o r s e s w h e t h e r an a t t e n u a t i o n o r a final inactivation o c c u r r e d , b e c a u s e of the mild subclinical infection t h a t m a y result f r o m such e x p e r i m e n t s . Nature of the

Virus

O n the basis of the histology a n d his studies related to the i n f e c t i o u s ness of the protein f r a c t i o n s as p r e s e n t in virulent h o r s e s e r u m , M o c s y ( 2 9 7 , 3 0 0 ) a s s u m e d that the inciting f a c t o r of i n f e c t i o u s a n e m i a is merely a "stimulating s u b s t a n c e " ( R e i z s t o f f ) f o r m e d within t h e b o d y of i n f e c t e d animals a n d that it is a nonliving p r o t e i n acting as a virus, b e i n g p r e s e n t in the a l b u m i n f r a c t i o n . H e c a m e to these c o n c l u s i o n s p a r t l y b e c a u s e the virus survived r e p e a t e d precipitation of p r o t e i n f r a c t i o n s w i t h o u t a decrease in its virulence a n d b e c a u s e it w a s n o t i n a c t i v a t e d b y i r r a d i a t i o n with X - r a y s . Balozet ( 2 5 ) o p p o s e d this c o n c e p t o n g r o u n d s t h a t t h e s u p p o r t i n g evidence w a s n o t s a t i s f a c t o r y ; h e believed t h a t t h e virus w a s simply a d s o r b e d to the p r o t e i n f r a c t i o n s tested by M o c s y , a n d t h a t t h e dosage of X - r a y used by him w a s t o o small. M a r t i n ( 2 7 9 ) d e m o n s t r a t e d that the virus is p r e s e n t in b o t h the a l b u m i n a n d t h e globulin f r a c t i o n s of infectious s e r u m . A t p r e s e n t the prevailing view is t h a t t h e virus of e q u i n e infectious a n e m i a is a filter passing, living m i c r o o r g a n i s m .

CHAPTER 6

METHODS

OF LABORATORY

DIAGNOSIS

Infectious anemia, in any of its stages, can by proper means be transmitted to normal horses for the purpose of confirming the diagnosis. Because of the expense involved, however, this method cannot be utilized as frequently as may be necessary. Therefore, efforts have been made to develop laboratory procedures, such as specific serologic tests or other methods, that could be used in making a decision in the practical diagnosis. The peculiar nature of the disease and the fact that the virus has not been cultivated so far are mainly responsible for the present limited success in developing diagnostic procedures that will meet all the requirements of a practical test, since it is not yet possible to overcome many of the difficulties involved. Nevertheless, there are a number of laboratory methods that can be used in combination either to support or disprove the clinical diagnosis of equine infectious anemia. The Problem of Specific Serologic

Diagnosis

Complement-fixation Test.—Many infectious diseases of bacterial or virus origin give rise to the occurrence of specific complement-fixing antibodies in the blood serum of the affected host. Such antibodies can, as a rule, be detected after the symptoms have been present for some time and usually last for a considerable time following the clinical recovery of the animal. In the case of equine infectious anemia, the latter observation should be of great value in identifying clinically recovered, symptomless virus carriers. As long ago as 1909 Hempel ( 1 6 0 ) reported that the complementfixation test is of no value in the diagnosis of equine anemia. Wirth ( 4 8 7 ) , using boiled extracts of clotted blood as antigen, also recorded negative results. Luhrs ( 2 6 7 ) , on the other hand, found that in a few cases of infectious anemia an extract of kidney, spleen, and liver furnished a suitable antigen that reacted with sera taken during the. fever reaction; cases in the afebrile stage gave negative results. Zeller ( 4 9 4 ) came to a similar conclusion, but noted that not all febrile cases react positively with good antigenic extracts. He also tried with inconclusive results the conglutination (31)

32

EQUINE INFECTIOUS A N E M I A

technique similar to the one reported by Schoening and first utilized by Pfeiler and Weber in the diagnosis of glanders. Mohler (303) prepared water extracts from the spleen of infectious anemia horses to be used as antigen, and published a preliminary note in 1936 reporting positive results in some cases. However, there seemed to be no regularity in his findings; the same animal that produced a suggestive positive result on serum from one bleeding, gave a completely negative reading at the next bleeding, and some of the proven cases of experimental infectious anemia never produced a positive reaction in the test, thus making the test useless for practical diagnosis. Traub and co-workers (458) hyperimmunized horses with 10 to 16 injections of infectious material and obtained positive results when testing their sera against suitable antigenic preparations (spleen extracts). They concluded, however, that the method cannot be used for practical diagnosis, since only one out of 25 field cases of infectious anemia gave positive result in the test. As part of the present studies here to be described, much time was spent in working out a suitable complement-fixation test by the use of modified and improved techniques. Some of the findings of earlier investigators were duplicated, but consistent and reproducible results were not obtained. At first, attention was mainly concentrated on preparing a suitable antigen from various organs and from the blood of infected horses.* The technique of the test proper was a somewhat modified complement-fixation test as described by Kelser and Schoening (201) for the serological diagnosis of glanders. Spleen and liver extracts often gave positive fixation with lower dilutions of sera from some of our experimental horses, but in general the experience was similar to the one recorded by Mohler: the results were inconsistent and transitory even in the same horse, and some animals failed to present a positive reading in the test during the whole course of their illness. An attempt was made to adsorb an eventual reacting antigenic component of infectious anemia blood to normal red blood cells of horses and also to chicken red blood cells at refrigerator temperature. This was then eluted at incubator temperature from the cells, centrifuged, and the clear supernatant used as an antigen against serum samples of normal, diseased, and seemingly recovered horses. Experimental antigenic preparations, kindly furnished by Doctors C. D. Stein and W. M. Mohler of the U. S. Bureau of Animal Industry and prepared by various methods of extracting red blood cells and plasma of infectious anemia horses, were also utilized. The results obtained in both groups, i.e., with eluted antigens and with the rest of the antigen preparations, were largely negative; some slightly positive readings noted in serum dilutions of 1:4 to 1:16 were not readily reproducible. * The first group of complement-fixation experiments were carried out by Dr. Werner Leemann of the University of Zurich, Switzerland, working in our laboratory.

6.

M E T H O D S OF LABORATORY DIAGNOSIS

33

Since a laboratory test for checking the results in other types of experiments, such as cultivation of the virus and its adaptation to laboratory animals, seemed to be essential for us, renewed efforts were made later in order to work out a procedure sufficiently sensitive at least for experimental purposes. For working with small amounts of serum and antigen, the technique described by Dreguss and Farkas ( 9 8 ) for serologic studies in typhus fever was adopted. In this method 0.2 cc. of each constituent is used (diluted serum, complement, antigen, and physiologic salt solution), giving a total volume of 0.8 cc. in the first phase of the test, before adding the hemolytic system. The sensitivity of the method was checked by an immune rabbit serum prepared by repeated injections of horse serum. The rabbit serum gave a definite four-plus fixation up to a dilution of 1:160 when tested against antigens of normal or infectious horse serum diluted 1:1200, or against suspensions (clear supernatants) made from organs of horses in dilutions of 1:200, while proper controls included in these tests were completely negative. Using this refined technique, a number of antigen preparations were tested against serum samples collected in all phases of the illness and also following recovery. Graded dilutions, rather than an empirically selected amount of the serum, were tested against antigens of various strength. Antigens were prepared from tissues of infected horses collected aseptically at the time of autopsy, with physiologic salt solution as the suspending material; the clear supernatant obtained after centrifugation was used in dilutions ranging from 1:5 to 1:200 to the original wet weight of organs (liver, spleen, kidney, lungs). Hemolysed red blood cells of normal and sick horses were tried as antigens; hemolysis was achieved by sterile distilled water, and the hemolysate was made isotonic by adding the proper amount of sodium chloride before use. Lung suspensions were included, as they often render good service as complement-fixing antigens in virus diseases. One of us (Dreguss ( 9 7 ) ) used saline suspensions of the lungs of mice infected with human and swine influenza virus for thousands of complement-fixation tests in studies related to influenza. Recently, Randall, McVickar and Doll ( 3 6 1 ) published a procedure of complement-fixation test for infectious abortion of mares, utilizing lung tissue of the affected horses as antigen. The advantage of lung tissue is that unspecific organ-fixation is less common with this type of antigen. Since none of these antigens proved to be satisfactory so far as a practical complement-fixation test is concerned, no detailed account will be given here. Although fixation to a lesser degree occurred with many of them, it is uncertain whether it was specific. The difference between normal and pathologic sera was mostly too slight to be sufficient for differentiation. A somewhat higher degree of fixation might have a theoretical significance, even though nonspecific. For instance, positive fixation with

34

EQUINE INFECTIOUS A N E M I A

liver antigens could be regarded as a sign of liver damage, although this might occur in many diseases other than infectious anemia. Such finding would indicate that the liver tissue became antigenic during the disease process and provoked the production of auto-antibodies. In this connection, following a technique similar to that of Bjorneboe and Krag (39) in their report of positive complement-fixation with liver TABLE 2 COMPLEMENT-FIXATION T E S T WITH VARIOUS ANTIGENS AND SERA

Antigen

Made of

Serum

Tested

Positive in Dilution

1:80

Lungs (Horse 2 1 )

Rabbit 392

Lungs (Horse 2 1 )

Rabbit 390

1:20

Lungs (Horse 2 1 )

Rabbit (20th transfer)

1:10

Red blood cells (normal horse) Lungs (passage mice)

Rabbit 392

1:40

Rabbit 22

1:50

Lungs (passage mice)

Rabbit 2 2 (normal)

1:10

Lungs (normal mice)

Rabbit 22

1:20

Lungs (normal mice)

Rabbit 21

(neg.)

Cultured virus (4th passage)

Horse 10 (16th day)

1:40

Cultured virus (4th passage)

Horse 10 (24th day)

1:20

Cultured virus (13th passage)

Horse 17 (20th day)

1:20

Cultured virus (13th passage)

Horse 17 (before inoc.)

1:10

Serum, Horse 20 (4th day)

Horse 2 (26th day)

1:160

Serum, Horse 20 (4th day)

Horse 17 (before inoc.)

1:40

cxtract antigen in cases of human hepatitis, we attempted to use liver suspensions of normal and infected horses as the antigen. However, these usually exhibited such a high degree of anticomplementary effect that they had to be greatly diluted, and this in turn made positive fixation unlikely. For example, a normal liver suspension in saline used as the antigen in a final dilution of 1:3200 produced all negative results with a number of infectious anemia serum samples taken during fever attacks or remissions; on the other hand, stronger liver suspensions could not be utilized as they were anticomplementary. Liver extracts were not tried. Some of the results in the complement-fixation tests are summarized in Table 2. Tests that gave negative readings, and antigens or sera exhibiting anticomplementary effect, are not listed in the summary of results.

6.

M E T H O D S OF LABORATORY DIAGNOSIS

35

As already mentioned, the positive findings in these tests were irregular and often inconsistent, and the titers were too low to be of practical value for diagnostic use. We made an attempt to bring about a reaction between acute phase sera, and sera collected in the later stages of the illness or during convalescence. Davis ( 7 6 ) and Hudson ( 1 7 7 ) have described a complementfixation test for yellow fever in which they used with satisfactory result acute phase serum of experimentally infected monkeys as the antigen against sera of humans and monkeys recovered from yellow fever infection. In our tests, using a similar technique, a slight suggestion of a positive reaction was noted. The serum of an infected horse taken on the 4th day after infection, and used as an antigen in a dilution of 1:5 and 1:10, evidenced positive fixation when tested against the serum of horses taken on the 26th to 42nd day of infection. The positive titers of the latter sera were as high as 1:80 to 1:160, and the results were reproducible. However, normal horse sera in lower dilutions also produced positive readings. It is known that some mammalian and avian sera, among them certain immune horse sera, do not fix complement in spite of the presence of the corresponding specific antigen. This applies, for example, to the reaction between pneumococcus antigens of carbohydrate nature and their antisera prepared in horses ( 3 6 5 ) . In such instances the so-called indirect complement-fixation can be used to disclose the presence of specific antibodies. Therefore, in addition to the tests performed with the usual (direct) technique, the indirect complement-fixation was utilized in our work in a few instances; however, there was no appreciable result. The technique followed was largely the one suggested by Rice ( 3 6 5 ) , and a simplified version as described by Wolfe and co-workers ( 4 9 1 ) for testing avian sera for Newcastle disease antibodies. In the application of these indirect methods for the test in infectious anemia, immune sera prepared by injection of materials containing horse protein cannot be used in the direct phase of the reaction, unless the antibody to horse protein is first removed. Immune sera obtained from our laboratory animals in attempts at transmission of the virus to them, produced surprising results in complement-fixation tests. Many serum samples of rabbits gave positive readings against antigens prepared from the lungs of infected horses. But the same sera also fixed complement with normal and infectious horse blood. These results were not attributable to antibodies to foreign protein (horse blood), since they were noted in tests with sera of rabbits that were injected with rabbit passage material during serial transfers of the virus in them. Similar positive results were recorded when organs, usually lungs, of presumably infected mice were set up as antigens against sera of infectious anemia horses. However, organs of normal mice also fixed complement to lower titers with the same sera. At the time when the experiments on complementfixation were performed, we regarded these findings as indicating unspecific fixation. But after reviewing all the evidence at present on hand, we are

36

EQUINE INFECTIOUS A N E M I A

inclined to believe now that the results as outlined above may have more significance than we at first suspected. We shall return to this question in the discussion below. The unsatisfactory results of complement-fixation tests recorded by other research workers as well as by ourselves may be explained in various ways. One explanation may well be that antigenic materials that were used in the experiments contained too low a concentration of the specific antigen, thus the latter did not reach the threshold value required for good results in any in vitro test. In general, viruses comparable in size to the virus of infectious anemia (e.g., poliomyelitis virus) do not readily supply antigens strong enough for complement-fixation tests. The inconsistency of slight fixation noted in blood samples of the same horse taken at different times can be best explained by assuming that the relative concentration of the antigen, and possibly also that of the specific antibody to the virus, varies from time to time. Therefore, the amounts are not always sufficient to be detected by complement-fixation. In certain phases of the illness, the antigen may be coupled and thus partly neutralized by the antibody, while at other times there may be more free antigen within the blood stream or in some of the organs of the infected animal. The behavior of the small laboratory animals' sera (particularly the sera of rabbits exhibiting a rather constant serologic response) and the results noted when testing, in combination, acute phase sera of horses and those collected during a later stage of the disease, require special consideration. It is conceivable that in the early phase of the disease an antigenic product is present in the blood of sick horses, probably derived from the erythrocytes attacked by the virus, to which antibodies are produced during the further course of the illness, and that this component has a common counterpart in the blood of other animal species. If so, any blood (or organs containing blood) might have the role of one reactive component in the chain of the serologic reaction. There are in our work indications, to be discussed later, that the course of events may be close to or at least compatible with this description. The working of the serologic test would be then somewhat analogous to the situation as seen in connection with human syphilis. As it is known, the "Wassermann antigen" used in the serologic diagnosis of that disease may be obtained from normal beef heart. If the seemingly conflicting results that we noted in some of the tests are based on a "tissue" antigen, the reaction may not be expected to be strictly specific. It is possible that more concentrated and, in particular, purified antigens may lead to a complement-fixation test at least for immunologic studies in the laboratory, even though its practical application for diagnosis of the disease or for identifying virus carriers might be limited. Cultivation of the virus offers the possibility of eventually preparing better antigens.

6.

M E T H O D S OF LABORATORY DIAGNOSIS

37

Moreover, it might be profitable to investigate further the use of lungs and lymph nodes of infected animals collected in the early, acute phase of the disease. Precipitin Test.—Hughes ( 1 7 8 ) reported that sera taken from monkeys or h u m a n s recently recovered from a severe yellow fever infection contain a precipitin capable of reacting with a precipitinogen present in the blood of monkeys during the acute phase of the illness. The concentration of the latter reflected the severity of the illness, although it was not considered identical with the virus of yellow fever. Adopting the technique of Hughes, we made some precipitin tests with sera of our experimental horses. Into a narrow serologic test tube, 0.2 cc. of the serum to be tested for the presence of precipitins was pipetted; then graded twofold dilutions of the acute phase serum (antigen) were prepared in saline, and 0.5 cc. of each dilution was carefully layered on the top of the test serum. Readings were taken after the tubes had stood in the incubator for 120 minutes. Formation of a ring-like precipitate at the contacting surface of the two layers was regarded as a positive reaction. Results were expressed in the highest dilution of antigen that would bring about the formation of a precipitate. Many combinations were tried. Some early serum samples gave good reactions, while others did not. Considerable difference was noted also in the behavior of sera from later bleedings tested against the same antigen. Many tests were performed in which a serum sample (Horse 4 ) taken at the peak of the first marked fever attack served as antigen. N o definite conclusions could be drawn from the positives in the tests. However, the impression gained was that a positive precipitin test would first occur about the time of the second fever attack. Titers ran as high as 1 : 1 2 8 0 or higher. In later stages of the disease the positivity declined. N o direct correlation was noted with the albumin-globulin ratio or the g a m m a globulin content of the sera tested. A number of false positive tests were seen. In some instances a precipitate would form at the juncture of the liquids upon the addition of saline instead of diluted antigen. Naturally, such sera gave a false positive reaction at any dilution of the antigen used for testing. This phenomenon was probably connected with the altered colloid stability of the sera in question. T h e sera of some horses (often the normal sample also) would give positive reactions from the very beginning of the experimental infection, when tested against an early phase serum. We were not able to discover the reason for these unspecific positive results. N o explanation of the precipitin test will be attempted here, as the data collected are not sufficient for an analysis. The preliminary work was discontinued because of the questionable value of the conflicting results.

38

EQUINE INFECTIOUS

Sedimentation

Rate of

ANEMIA

Erythrocytes

The sedimentation rate of the red blood corpuscles was suggested for diagnostic purposes in infectious anemia by Noltze ( 3 2 3 ) . He adapted the technique used in connection with human diseases to larger amounts of horse blood (25 cc.) and concluded that an evenly accelerated settling out of red cells is diagnostic, or at least highly suggestive of infectious anemia. Kuhn ( 2 2 8 ) and Luhrs ( 2 6 8 ) disputed Nolze's conclusions on the grounds that several other diseases may produce the same phenomenon, while some infectious anemia cases, particularly during remissions, would not show an accelerated sedimentation. Schermer and co-workers ( 3 8 7 ) , on the other hand, considered the test as a useful aid, if used in combination with hematologic tests and protein determinations. Later, many authors contributed to a proper evaluation of the test (149,234,251,293, 321,330,343). In this country, Hammersland and co-workers ( 1 5 0 ) and also Griffin and Brose reported on increased sedimentation rates in infectious anemia. The former investigators utilized Cutler's technique and noted on citrated blood an average sedimentation of 18 mm. in normal horses, while infectious anemia cases showed an average of 26 mm. On account of the different technique they used, their values cannot be compared with ours. Since many factors including the anticoagulant, the instruments used, and the timing of reading the results, etc., exert a definite influence on the numerical values recorded in the sedimentation test, only such results as were obtained with identical techniques can be compared (Nichols). Gilnian ( 1 3 5 ) , who investigated the sedimentation rate in this laboratory on our experimental horses, used oxalated blood samples and tested them within two hours of collection under strictly uniform conditions by a somewhat modified Westergren technique, which is widely employed for studying the sedimentation in human diseases. Readings were made at 10, 15, 20, 30, 45, and 60 minutes, recording the fall of erythrocytes in millimeters. The sedimentation rate was compared with the hematocrit values and it was found that in acute cases there is a fairly good correlation between the two. Increase of the sedimentation rate largely paralleled the drop in the red cell volume. When the mean sedimentation rate per ten minutes ( S R / 1 0 ) was calculated according to van Zijl from the readings at 30 and 60 minutes and the values were corrected for an average red cell volume of 42 per cent, no significant changes were found in these calculated figures during the course of the acute stage of the disease. From this Gilman concluded ( 1 3 5 ) that the changes in sedimentation rate in this particular phase of the disease were largely due to changes in red cell volume. According to the present concept of the nature of increased sedimentation rate, alterations in the plasma proteins are the main factors responsible for the phenomenon, particularly the increase in the fibrinogen

6.

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39

DIAGNOSIS

and globulin components. These changes seem to run parallel with the decrease of the red cell volume (hematocrit) in acute infectious anemia. In general, our findings in connection with the changes in the sedimentation rate during the course of infectious anemia suggest that the readings can be used (without correction), in conjunction with the clinical diagnosis, for judging the activity of the infection prognostically. But the behavior of the sedimentation rate can neither prove, nor rule out a diagnosis. We shall here summarize the observations on general trend as seen in our experimental horses. TABLE 3 S E D I M E N T A T I O N R A T E O F E R Y T H R O C Y T E S IN I N F E C T E D H O R S E S

Davs foil. infection (before) 4 6 11 16 20 24 30 36 42 50

Horse 29 (acute Temp. F°

100.0 102.2 99.8 104.8

R.B.C. mill.

10

7.8 7.1 7.4 5.6 5.8

2 5 3 13 21

Horse 22 (subacute

case)

Sedimentation in 20 JO 60 minutes 11 31 29 55 95

33 64 65 96 Ì118

88 105 108 125 125

Temp. F° 99.4 99.6 99.2 103.4 99.0 101.2 101.4 103.4 104.4 104.0 103.0

R.B.C. mill. 8.6 9.0 9.1 7.1 6.3 7.6 7.4 6.6 6.5 6.8 4.5

case)

Sedimentation 10 20 30 minutes

in 60

9 47 33 92 65 38 57 43 22 35 97

55 86 96 111 104 89 89 90 74 85 127

3 3 4 26 26 13 30 14 6 14 28

19 73 76 103 83 60 72 61 42 54 113

Individual variation of the sedimentation rate in normal horses covers a fairly wide range; therefore, no strictly valid figures can be given for all horses. The sedimentation is much more rapid in horse blood than in that of most domestic animals or of humans. In 10 minutes, values are reached with horse blood equaling those noted at about 60 minutes with human blood. By using the above-mentioned method, the large majority of normal horses presented a sedimentation rate of 2 to 12 mm. in readings at the end of 10 minutes, while in 20 minutes the range was from 10 to 60 mm., and at 60 minutes between 15 and 90 mm. If extreme values are noted temporarily in a seemingly normal horse, the animal must be considered as not in normal condition. The sedimentation rate in our infected horses varied considerably. Generally, during or even before the first marked fever attack, the sedimentation rate would increase and readings from about 20 to 50 would be noted in 10 minutes, while after 30 minutes the range was between 50 and 130 mm. These were the readings most often observed, but deviations were not infrequent. Table 3 illustrates some of the readings

40

EQUINE INFECTIOUS A N E M I A

obtained in an acute ( H o r s e 2 9 ) a n d a subacute case ( H o r s e 2 2 ) during a typical course of experimental infectious anemia. H o r s e 29 was observed f o r a total of 18 days following infection. T h e figures as shown in t h e table represent direct readings expressed in millimeters. T h e extensive investigations carried out on our horses indicate that in cases of longer duration no strict rules seem to exist. In some of the chronic cases the sedimentation rate would remain in a b o u t the n o r m a l range even during fever attacks. T h e same applied also to the horses that failed to exhibit anemia following challenge inoculation. Others would show a definite increase in the sedimentation rate early in the course of the disease and, in spite of an a p p a r e n t recovery, the s e d i m e n t a tion rate would remain at a relatively high level suggesting that the p a t h o logic condition of the animal did not sufficiently balance in spite of seemingly improved clinical health. Such animals would, sooner or later, experience an unexpected flare up of symptoms. T a k i n g our results as a whole, it may be said that an increased sedimentation rate is a regular feature in acute and subacute cases; not so, however, in the chronic stage of the disease, when the sedimentation rate either would run largely parallel to the activity of the infection or would r e m a i n within or close to the normal range in spite of clinical symptoms, as mentioned already. We were not able to determine in our experimental animals w h e t h e r the sedimentation rate would increase u n d e r strenuous l a b o r in chronic cases or virus carriers, because our horses were not w o r k e d or exercised. Attempts have been m a d e in the past to use the sedimentation test f o r screening herds in order to eliminate latent infectious anemia cases. H o w ever, it must be r e m e m b e r e d that there have been reports ( 1 4 9 , 2 3 4 , 3 8 5 ) , confirmed by our investigations, that the sedimentation test often fails, particularly during remissions and in asymptomatic virus carriers. In view of the fact that also other disease conditions m a y show an increased sedimentation rate of the erythrocytes, the test is not well suited to decide alone the differential diagnosis in infectious a n e m i a ( 2 2 8 , 2 6 8 ) . According to L a m a r r e ( 2 3 4 ) , Panisset ( 3 4 3 ) , and O p p e r m a n n ( 3 3 0 ) , the sedimentation can be used for differentiating infectious and parasitic anemia. This is apparently not always the case. Schermer and c o - w o r k e r s ( 3 8 5 , 3 8 7 ) stated that accelerated sedimentation may be observed in parasitic a n e m i a as well, but in the latter condition no relative lymphocytosis is present, while it is often noted in subacute or chronic infectious anemia. Lemetayer (251 ) pointed out that serum horses exhibited a definite increase of sedimentation rates following injections of antigenic material to them during the process of hyperimmunization. This observation fits well into the present concept about the mechanism of accelerated sedimentation of corpuscular elements, which is generally believed to be the result of alterations in the relative concentration of p l a s m a proteins, causing the erythrocytes to clump and settle faster than u n d e r n o r m a l conditions.

6.

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41

The findings of Knisely and his collaborators ( 8 1 0 ) tend to show that an increased sedimentation in vitro is not equivalent to agglutination in vivo. Intravascular agglutination of the latter type (sludged blood) was demonstrated by them experimentally in certain pathologic conditions ( 3 3 , 2 0 7 ) . It is conceivable, that a similar in vivo hemagglutination takes place temporarily in certain phases of infectious anemia. (See Chapter 8.) In our opinion, an increased sedimentation of the erythrocytes cannot be considered a diagnostic test for infectious anemia, but it might add pertinent information in diagnosed cases as to the status of the infection. It may be used for supporting the clinical diagnosis in early cases, provided other conditions, which may produce a similar increase, can be ruled out. Colloid Stability

Tests

The pathologic condition of a horse suffering from infectious anemia is reflected in qualitative and quantitative alterations of the colloids present in the liquid portion of its blood. The changes which occur in the relative amount of the protein fractions, lipids, etc., exert an influence on the stability of the various colloids suspended in the blood fluid. Several authors have attempted to use these pathologic changes of the blood serum or plasma as an aid in the diagnosis of infectious anemia. It is to be assumed that none of them would be specific; nevertheless they may serve as supporting evidence for confirming the diagnosis. A great number of colloid stability tests were suggested for use, among them the so-called sublimate test, the dependability of which was widely discussed. Tests based on the lipid content of infectious horse serum were suggested by several workers. However, Richters ( 3 6 8 ) stated that a Meinicketype lipid binding test gave positive reaction in some cases of infectious anemia, but it was not considered specific. Mocsy ( 2 9 6 ) compared as many as eight stability tests and noted that none of them gave results reliable for practical diagnosis. Wittfogel ( 4 8 9 ) and also Member and coworkers ( 2 8 5 ) found elevated cholesterol values during fever attacks in infectious anemia. As a contrast to this, the cholesterol content was found to decrease during the hyperimmunization of serum production horses (Lemetayer ( 2 5 1 ) ) . Boshyan ( 4 2 ) concluded that while the protein level decreased, the nucleic acid and polypeptid content of the plasma increased during fever attacks. On this ground he devised a colorimetric test that was supposedly 90 per cent accurate in detecting infectious anemia cases. His findings were not confirmed and the results were not duplicated by others. Further tests which were suggested for use in infectious anemia are the inhibition test of saponin-hemolysis (Abderhalden and Frei ( 2 ) ) , and the formolgelatine test. LUhrs ( 2 6 4 ) , Lehnert ( 2 4 4 ) , and Slagsvold ( 4 1 2 ) stated that the saponin test is of little value for diagnostic purposes, while Wittfogel

42

EQUINE INFECTIOUS

ANEMIA

( 4 9 0 ) concluded that the formol-gelatine test can be used as an aid in diagnosis, but it is not specific. Although based on the serum colloids, the colorimetric test and the saponin-hemolysis test are not devised to p r o b e the stability. Recently, L e e m a n n ( 2 4 2 ) described a simple test in which c a d m i u m sulfate solution is used for studying the colloid stability of horse serum. Based u p o n a similar test used in h u m a n medicine, it was considered by L e e m a n n as a supplementary aid in the diagnosis of suspected infectious anemia cases. A n o t h e r method often used to investigate the colloid stability is the Fulton test. Originally devised by Bennett and Kenny f o r the diagnosis of trypanosomiasis in camels, Fulton ( 1 2 9 ) adapted it f o r use in infectious anemia. According to him the serum of healthy horses gives a precipitation, with solutions of mercuric chloride (sublimate) in dilutions of 1 : 2 0 , 0 0 0 , but not with weaker solutions. O n the other h a n d , the serum of horses afflicted with equine infectious anemia gave a positive reaction with dilutions of mercuric chloride up to 1 : 1 0 0 , 0 0 0 . T h e mechanism of the reaction is based on alterations in the serum proteins. T h e value of the test as a diagnostic aid has since been considered by a n u m b e r of authors, as Urbain et al. ( 4 6 4 ) , Lemetayer ( 2 4 9 ) , O p p e r m a n n ( 3 2 9 ) , H e c k e ( 1 5 6 ) , Hillmer ( 1 6 2 ) , H a m e r s k i ( 1 4 9 ) , Derrick ( 8 0 ) , and others. M a n y c o n sidered the reaction nonspecific, or misleading ( 8 0 , 1 5 6 , 1 6 2 ) ; some infectious anemia horses have been shown to give a negative reaction ( 1 4 9 , 1 5 6 , 3 2 9 ) , usually the symptomless carriers. O n the other hand, positive reactions have been observed in clinically healthy horses ( 4 6 4 ) , in horses h y p e r immunized to produce therapeutic sera ( 2 4 9 ) , and in horses with equine encephalomyelitis ( 8 0 ) and strangles ( 4 6 4 ) . In cases of parasitic a n e m i a the test was found negative ( 4 6 4 ) . As expected, none of the serum colloid stability tests can be considered specific for equine infectious anemia. Nevertheless, they may furnish information of value for proper critical evaluation of other data. Albumin-Globulin

Ratio of Blood

Serum

Since it is known from the histopathologic findings that the liver is seriously affected in infectious anemia, we conducted a detailed study of the blood proteins because of the role the liver plays in the protein m e t a b o lism. T h e relative a m o u n t s of the two main groups of proteins present in the serum were determined in the experimental horses. It was expected that the determination of the albumin-globulin ratio would be helpful in the diagnosis, as it is in some h u m a n diseases, and might furnish useful information concerning the disease process, particularly with regard to the development of debility, emaciation, and edema.

6.

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DIAGNOSIS

43

It has been known for some time that blood proteins undergo changes and globulins increase during the disease ( 2 4 4 , 3 8 1 ) . Schermer ( 3 8 4 ) suggested that the hyperglobulinemia can be used, along with the hematologic findings, as a criterion in the differential diagnosis. In two carefully studied cases of infectious anemia he noted globulin values of over 80 per cent of the total protein, as against the normal figure of approximately 50 per cent. He probably used Howe's method for determination of the globulins, which gives a higher albumin-globulin ratio than is obtained with more recent methods. Howe's technique applied 23 per cent sodium sulfate concentration for salting out the globulins and it was in general use until recently. Careful comparison of the chemical and physico-chemical methods has shown that a practically complete precipitation of the globulins can be achieved only at a 26 per cent saturation with sodium sulfate; this will furnish results that are in fairly close agreement with those obtained by electrophoresis analysis ( 2 0 3 ) . Using such recently developed methods, we obtained values of serum protein fractions comparable to those given by electrophoresis ( 8 1 ) ; these deviate significantly from the figures recorded by the use of older techniques ( 7 3 , 3 8 1 , 3 8 4 ) , working with 23 per cent sodium sulfate. Part of the findings, mainly the ones noted in acute cases of infectious anemia, have been published from this laboratory by Gilman ( 1 3 6 ) with a detailed description of the methods. In the studies on which our present report is based, the total protein was determined by a slightly modified Greenberg procedure ( 6 9 0 ) through the addition of a 10 per cent sodium hydroxide solution and Folin's phenol reagent to the properly diluted horse serum; the blue color thus developed was read in a Klett-Summerson photoelectric colorimeter standardized by the use of known tyrosine solutions. For determination of the serum albumin, the globulins were precipitated at 26 per cent sodium sulfate saturation and filtered, both at incubator temperature. The amount of albumin in the clear filtrate was then determined in the colorimeter after the addition of Folin's phenol reagent and sodium hydroxide as above. The globulin was calculated by subtracting the value of albumin from that of the total protein. The proportion of albumin to globulin was calculated and expressed as A / G , that is, albuminglobulin ratio ( 1 3 6 ) . Recent data taken from the literature ( 8 1 , 8 2 ) , as well as our own results, indicate that the globulin fraction constitutes the greater part of serum proteins in normal horse blood. In normal horses with a total serum protein of 6.0 to 8.5 grams per 100 cc. the globulin fraction amounts to about 4 to 5 grams per 100 cc. that is approximately twice the figure given for albumin. The normal A/G ratio in our horses before experimental infection as well as in a group of other healthy horses generally ranged between 0.5 and 0.8. In a few instances values of 0.8 to 1.0 were noted.

44

EQUINE INFECTIOUS

ANEMIA

A detailed analysis of the results f r o m m o r e than 4 0 0 serum samples of experimentally infected horses is not w a r r a n t e d ; instead, a brief s u m m a r y of the pertinent findings will be presented. O i l m a n ( 1 3 6 ) published an account of several acute cases in our series. It m a y be said generally that a significant decrease of the albumin-globulin ratio was observed in the serum of infected horses during the acute and subacute stage. This was brought about by a primary drop of the albumin level, closely followed by a m a r k e d rise in the globulin fraction. A slight decline in the ratio may be noted as early as the 9th or 10th day following infection, then, as a rule, a very marked decrease occurs between the 14th and 2 3 r d day of infection. T h e lowered ratio is maintained through the active p h a s e of the disease. As the illness progresses, the A/G ratio progressively declines. In the majority of our horses infected f o r passage of the virus, a decreased ratio of 0.45 to 0 . 2 0 was f o u n d within the first three weeks of infection, and values as low as 0 . 1 2 or 0 . 1 0 were seen in some cases. Such horses manifested debility and considerable loss of weight. In chronic cases the ratio either fluctuated at a somewhat decreased level or r e t u r n e d to the lower normal range, with only slight drops f r o m time to time coincident with the relapses. A well-balanced, symptomless carrier p r o d u c e d A/G values which did not differ f r o m those obtained in n o r m a l horses. During the experimental immunization with organ suspensions, an early decline of the A/G ratio was noted in one animal ( H o r s e 2 5 ) ; w h e n challenge inoculation was attempted, this horse first exhibited a transient increase in the ratio followed later by a m a r k e d drop. A n o t h e r animal similarly treated ( H o r s e 3 0 ) did not show a decreased A/G ratio w h e n receiving the first few doses of vaccine: but when the horse received the second group of immunizing injections and following challenge inoculation the ratio dropped. It is not certain whether the early reactions were d u e to eventual subclinical infection by the vaccine used, or to the vaccine as such having provoked the production of antibody globulins. It may be added that egg culture materials, p r e s u m a b l y containing infectious anemia virus ( C h a p t e r 1 1 ) , only slightly influenced the A/G ratio in the serum of recipient horses. O n the other h a n d , virus that was carried in serial mouse passages, when injected to horses, b r o u g h t a b o u t a marked drop of the ratio from 0.61 to 0 . 3 5 in 22 days ( H o r s e 1 0 ) , a n d from 0.47 to 0.29 in 39 days ( H o r s e 1 5 ) , respectively. As already mentioned, the numerical values of albumin-globulin ratio recorded in the literature were obtained with a different technique, t h e r e f o r e they are not directly c o m p a r a b l e with our results; but they indicate the same general trend. Schermer ( 3 8 1 ) noted a physiologic increase in the relative a m o u n t of globulins in hard working horses; other horses o n a starvation diet also showed a relative globulin increase in their sera, while the total protein remained within normal limits ( m e a n i n g a -decrease in the A/G r a t i o ) , but the alteration was not as m a r k e d as in infectious

6.

METHODS OF LABORATORY DIAGNOSIS

45

anemia. Furthermore, he found that in chronic infectious anemia cases the relative percentage of the globulin fraction was, in average, one and one-half times higher than the average in 30 normal horses. It is conceivable that infection, hard work, and poor diet may act together in some field cases to produce an extremely low value of A/G ratio. The diagnostic value of the albumin-globulin ratio is, naturally, limited because of the close to normal values often recorded in chronic cases. On the other hand, a marked decrease of the ratio during the acute and subacute stage is quite regularly observed, and seems to be an integral part of infectious anemia. It must be kept in mind, however, that similar changes occur also in other conditions associated with liver damage. Liver Function Tests Serious impairment of the liver function can be detected by liver function tests performed on the serum of patients. A great selection of such tests are used in human medicine ( 4 8 ) , but they cannot be applied to horse serum without adaptation because of the differences in the composition of human and horse blood. The various liver function tests that yield information concerning certain types of hepatic lesion do not, as a rule, disclose early and mild pathologic changes, since the liver can maintain its function with only a small part of relatively intact liver tissue and also because of the great regenerative capacity of the liver. Thus, some of the liver function tests, if definitely positive, usually indicate an extensive liver damage. Within the scope of the present studies, an attempt was made to use two types of liver function tests. 1) Among the precipitation tests, the thymol turbidity test was selected. When positive, this was found to indicate very severe liver damage ( 1 3 6 ) . 2) One of the flocculation tests, Hanger's cephalin-cholesterol test ( 1 5 1 ) , was studied in some detail; it gave satisfactory results, after adaptation for testing horse sera, considerably more diluted than in the case of human serum, and if a suitable pH was maintained ( 1 3 7 ) . By testing a large number of normal and infectious anemia sera in this laboratory, Gilman ( 1 3 7 ) observed that the modified cephalin-cholesterol test became positive between the first and second marked fever attacks and usually remained so during the acute, subacute, and chronic phase of the experimentally induced disease, indicating that an impairment of the liver function is a regular part of the infectious process. The delicate test, the reading of which requires some experience, was regarded positive when three- or four-plus flocculation occurred in a dilution of 1:85 or higher. The dilution is expressed in a relationship of the amount of serum to the cephalin-cholesterol emulsion present in each test tube.

46

EQUINE INFECTIOUS

ANEMIA

Both, the thymol turbidity test and the cephalin-cholesterol flocculation test, are known to indicate parenchymal liver damage in h u m a n s ( 4 8 , 2 0 4 ) . T h e latter seems to be the more sensitive of the two when used as suggested for horse serum ( 1 3 6 , 1 3 7 ) . It is noteworthy that it gave consistently positive results in one of our experimentally infected horses ( H o r s e 10, a chronic case) that, although suffering a few relapses, was in good health for long periods of time. T h e liver was apparently so m u c h damaged that its function remained impaired. It remains to be seen by f u t u r e studies whether balanced, symptomless carriers (with no relapses) would behave in the same way. If so, the test may help in picking out suspect virus carriers from a group of horses, provided that other chronic conditions associated with liver damage can be ruled out. Obviously, neither of these liver function tests can be considered as specific for infectious anemia. Nevertheless, they can contribute to following the course of the disease and understanding the mechanism of the infection. Various Chemical Components

of the Blood-

Chemical constituents of the blood other than proteins were also studied by several investigators, but none of them revealed changes of diagnostic significance. Wright ( 4 9 3 ) found the iron content of blood and spleen decreased. Hammersland and co-workers ( 1 5 0 ) determined t h e urea nitrogen, dextrose, chloride, calcium, phosphorus, and iron content of the blood serum in infectious anemia and compared the values with those of normal horses. T h e average values showed a significant difference only in the iron and calcium content. In the infected group, iron was lowered while the calcium was elevated. T h e f o r m e r probably has some relationship to hemosiderosis in various organs, while the latter can b e explained by changes in the blood proteins and their inter-relationship to calcium and phosphorus content as described by Craige and G a d d ( 7 3 ) . The average values for blood sugar were somewhat diminished in the anemia group ( 1 5 0 ) . An apparently detailed study on chemistry and physico-chemistry of the serum in infectious anemia was published by Lay ( 2 4 0 ) , but it was not accessible to us either in original, or in abstract form. In our work, calcium determinations did not reveal any unusual changes. The same was true for attempts to follow non-protein nitrogen and blood sugar values. The sodium content of serum and plasma was extensively studied by means of a volumetric method described by Dreguss ( 9 1 ) . but aside from some decrease at the final stage of the disease, n o alterations beyond the limit of physiological fluctuation were noted in the sodium level.

6.

M E T H O D S OF LABORATORY DIAGNOSIS

47

*

Although there is as yet no specific test for the laboratory diagnosis of infectious anemia, a combination of several tests may render a good service in the diagnosis, if considered in conjunction with a careful clinical observation and repeated hematologic investigations. Histopathologic findings, post mortem, can add further data of diagnostic significance, although they are not specific, and liver biopsy may serve to follow the evolution of characteristic pathologic alterations in the liver, while the animal is alive. Methods of laboratory diagnosis, which were not dealt with here ( H e m a tology, Hemagglutination, and Histopathology) are discussed later in subsequent chapters.

CHAPTER

7

HEMATOLOGY Descriptions of equine infectious anemia contain many contradictory statements on hematologic findings noted in the condition. This is partly due to the fact that various authors studied the changes of the blood in different stages of the disease and did not take into consideration that the blood picture is constantly changing during the course of the infectious process reflecting, to a certain extent, the systemic response of the organism to the infection. Another possible source of the controversy may well be that the unusual hematologic findings were noted either in anemic horses suffering from mixed infections, or in sick animals with no satisfactory evidence that the anemia observed was in fact caused by the virus of infectious anemia. With the hope of clarifying some of the controversy, and also of collecting basic information about the nature of anemia in this disease, we regularly followed the hematologic changes in most of our experimental horses. Altogether over 600 bleedings were done. In one group, bleedings were performed when required by the particular experiment in progress. In a second group, devoted to a closer study of the hematology, blood samples were taken every second to fourth day or at about weekly intervals. We shall briefly summarize our findings here, then relate them to those described by other workers, and finally analyze the relative value and eventual practical use of the conclusions derived from these studies. Gilman ( 1 3 4 ) has published from this laboratory hematologic observations in the acute phase of the disease. In the present analysis based on the total of our data, part of which was collected by Dr. Gilman while he p a r ticipated in the experimental work, we shall discuss, in addition to findings in the acute stage, those noted in subacute and chronic cases of longer duration. Methods.—The horses were inoculated with the New H a m p s h i r e strain of equine infectious anemia virus. Of the total n u m b e r of horses used by us for experimentation, complete hematologic studies were p e r f o r m e d in twenty animals. Three of these will not be considered here because an interpretation of their values is difficult at this time owing to the type of experiments performed upon them. T h e rest of the horses received virulent (48)

7.

49

HEMATOLOGY

virus, infectious plasma or serum, taken at various stages from our passage animals. The technique for collecting blood and for making the differential blood smears, total red and white cell counts, hemoglobin and hematocrit determinations, and also the methods of calculating the mean corpuscular hemoglobin, and mean corpuscular volume were identical or similar to those which Gilman ( 1 3 4 ) referred to and which are in general use in such studies. Total serum protein was determined by a modified Greenberg procedure ( 6 9 0 ) as mentioned in the previous chapter on Laboratory Diagnosis ( p . 4 3 ) . Determinations performed on the blood of individual TABLE

4

H E M A T O L O G I C R E S P O N S E OF AN A C U T E C A S E ( H O R S E 2 9 )

Davs foil inoc.

Temp. F°

Total Serum Protein

R.B.C. million

W.B.C. thous.

(before)

100.2

6.81

7.78

8.55

59

30

2

4

4 5 6 9 11 15 16 18

100.0 101.6 102.2 100.0 99.8 103.2 104.8 105.4

6.58

7.05

6.50

49

35

4

4

6.90

7.35

6.95

48

35

7

5

6.84

5.58

7.40

73

19

2

1

7.86 7.98

5.79 4.51

5.95 7.60

59 47

20 39

1 3

3 0

Neutr. Lymph. per

Mono. cent

Eos.

animals prior to infection were used as controls, in addition to a number of other normal horses tested. The results of the hematologic investigations will be presented in two groups, those collected on changes in the red blood corpuscles, and the ones related to alterations in the number and relative percentage of the various types of white blood cells. The findings will be grouped according to changes noted in the acute, subacute, and chronic phases of the disease. Red Blood

Corpuscles

Acute Phase.—There is a primary fall in the number of erythrocytes between the second and fifth day after inoculation (Tables 4 and 7 ) . Recovery of approximately one-half of this loss occurs within the next few days. Between the tenth and sixteenth days there is a further fall in the number of red blood corpuscles that usually is accompanied by a marked temperature rise, which in most cases ranges from 102 to 106°F. (Tables 4 and 7 ) . At the time of the first fever attack the average decrease of

E Q U I N E INFECTIOUS A N E M I A

50

erythrocytes per cubic millimeter is about 2 to 2 and one-half million. In the interim between the first and second fever attacks, a gradual increase in red cells ensues and the greater part of the original normal value is often attained (Table 7 ) . During the first and second attacks, there is no apparent change in staining qualities, size, or shape of the erythrocytes. After TABLE

5

HEMATOLOGIC R E S P O N S E OF A SUBACUTE CASE ( H O R S E

22)

Days foil. inoc.

Temp. F°

Total Serum Protein

R.B.C, million

W.B.C. thons.

(before)

99.4

7.64

8.00

10.02

64

27

3

3

99.6 99.4 99.2 99.8 99.6 104.2 103.6 102.4 104.0 99.0 100.6 101.2 101.4 103.4 102.0 104.4 104.0 103.6 103.4 103.0

8.16 9.46 7.60 7.52 6.96 7.26 7.12 7.36 7.36 7.44 7.04 8.16 7.92 7.52 7.84 7.84 6.64 7.04 7.68 8.64

8.88 9.04 9.09 8.04 8.64 7.14 8.61 7.51 7.50 6.27 6.95 7.58 7.41 6.59 6.56 6.53 7.20 6.75 4.53 4.54

5.80 7.50 10.50 8.75 7.85 6.70 8.95 7.80 8.15 7.50 8.60 7.40 14.50 9.05 11.40 10.90 9.00 5.60 7.85 8.20

58 59 55 56 57 64 73 80 67 60 62 53 68 76 39 70 69 67 62 71

30 36 38 36 28 28 20 13 22 24 25 30 21 19 45 15 25 19 28 22

6 3 4 7 7 3 6 4 3 4 2 8 2 3 9 12 4 8 5 3

3 2 2 0 5 3 0 0 0 1 0 0 0 0 1 0 0 0 0 1

3 5 6 8 9 10 12 13 15 16 17 19 24 29 33 36 42 44 50 60

Neutr. Lymph. per

Mono, Eos. cent

three or more severe exacerbations, as the case tends to become subacute in nature, a few immature forms, usually normoblasts, are seen. Table 4 illustrates the findings in a typical acute case during the first 18 days following infection. The temperature was taken twice daily, but only the higher daily reading is given. Horse 29 was injected intravenously with 1 cc. of infectious serum from Horse 25 on April 28, 1950. On the fifth day following infection a mild temperature rise occurred, and a marked fever reaction began on the 13th day; the temperature reached 105.4°F. on the 18th day. At this time the animal was destroyed for another experimental purpose. As may be seen from the table, at the time of the mild

7.

HEMATOLOGY

51

temperature rise a slight decrease occurred and by the 11th day the erythrocytes decreased by two million as compared with the normal value. A week later the loss of red blood corpuscles exceeded three million. Subacute Phase.—When fever attacks occur at longer intervals and are less severe in nature, the loss of red cells proportionately diminishes (Table 7. The total serum protein, although varying little at the fever peaks, exhibits an increase between attacks (Table 6 ) . In the instances in which the fever attacks are marked, a. further diminution of the total red cell count occurs at each ensuing fever attack, with less and less reparation (Tables 5 and 6 ) . TABLE

6

H E M A T O L O G I C R E S P O N S E OF H O R S E 3 3

foil. inoc. (before)

Temp. F°

Serum Protein

R

B C

miUion

- W.B.C. thous.

99.6

7.66

6.71

8.60

HemogL (gram)

Hemalocrit

13.8

40

(SUBACUTE C A S E )

Neutr.

59

Lymph. per

Mono. cent

Eos.

35

1

5

14

105.6

7.84

4.62

4.95

9.2

24

61

34

1

1

24

99.2

8.05

5.82

9.65

11.6

33

60

32

5

3

27

106.0

7.79

4.46

7.85

10.2

28

67

20

4

1

34

100.9

8.16

3.71

5.90

7.7

22

41

50

5

1

41

101.4

7.34

3.20

8.70

6.3

18

41

49

8

2

65

105.0

7.84

1.41

3.80

3.0

8

60

31

6

0

72

99.8

8.30

2.60

10.85

5.2

15

45

40

11

0

Table 5 demonstrates the values in an experimental horse that was under observation for 60 days after it had received a single inoculation of 1 cc. of virulent virus (infectious serum). It produced three typical fever attacks; the third started on the 36th day following infection and the temperature did not drop below 103°F. until the horse was destroyed on the 60th day. Although the animal exhibited an initial rise and a definite decrease of red blood cells by the 16th day, it did not develop a marked anemia until the 50th day. The values presented in Table 6 are those recorded after another animal (Horse 3 3 ) received a challenge inoculation with a known virulent virus (5 cc. serum). This animal was previously injected twice for experimental purposes with serum pools, but did not show a clinical reaction. After the challenge inoculation the horse had several marked fever attacks usually exceeding 105 °F., as may be observed from Table 6. On the 64th day following infection, epistaxis occurred and continued until the horse was destroyed.

52

EQUINE INFECTIOUS A N E M I A

Chronic Phase.—As the febrile periods are less frequent and as they diminish in severity, the erythrocyte count often falls only slightly below the normal and the counts between attacks are in most instances within the normal range (Table 7 ) . Running largely parallel with the changes in the n u m b e r of erythrocytes, alterations can be noted in the total volume of red blood corpuscles (hematocrit) as determined by strong centrifugation of fresh whole blood after addition of anticoagulants. The hemoglobin content of the blood likewise exhibits marked fluctuations. Some early investigators of infectious anemia laid particular emphasis onto the proportionate or disproportionate diminution of hemoglobin content as compared with the decrease of the number of erythrocytes; some of these authors ascribed diagnostic value to the behavior of the relative amount of hemoglobin. However, we were unable to confirm this in our experimental horses. W e shall summarize below our findings related to hematocrit and hemoglobin values as noted in infected horses. Hematocrit.—Concurrent with the first fall in the n u m b e r of erythrocytes and hemoglobin, taking place during the first week of the infection, the average hematocrit value drops 6 to 8 per cent and improves in the following days, only to diminish markedly at the onset of the first m a r k e d fever attack. In subsequent attacks, a similar pattern is discerned. T h e hematocrit value at the time of the first attack averaged 29 per cent, whereas the normal average before inoculation was 41 per cent in 15 of our horses (Table 7 ) . During remissions the hematocrit value increases. Studies dealing with sedimentation rate of the red blood corpuscles, as determined under, controlled laboratory conditions on spontaneously sedimenting blood of infected horses, are discussed under methods of laboratory diagnosis (Chapter 6 ) . Hemoglobin.—In acute phase there is a primary decrease in h e m o globin content between the second and fifth day after inoculation, with an average loss of 1 to 2 grams in 100 cc. of blood. In the next few days there is an average gain of approximately 1 gram. Between the tenth and the sixteenth day of the experimental infection, there is an additional loss of hemoglobin (average 3 g r a m s ) . In the period between temperature rises, the hemoglobin value increases until it reaches the lower normal range (Table 7 ) . In the majority of our cases, a decrease in hemoglobin content, a fall in the erythrocyte count, and a temperature rise occurred simultaneously. Mean Corpuscular Hemoglobin, Mean Corpuscular Volume.—When these values were calculated, no significant changes were noted in o u r experimental horses during the acute phase of the disease, as stated by Gilman ( 1 3 4 ) . With the disease progressing, the rate of corpuscular h e m o globin may increase, evidently to compensate somewhat for the loss of erythrocytes and to promote oxygen transport needed for respiration and

7.

53

HEMATOLOGY

metabolism of the tissues. Such a need particularly exists in animals working or being exercised. White

Blood

Cells

During the first few fever reactions, the total number of white blood cells decreases, exhibiting values in the lower normal range or a definite leukopenia. The decrease is manifested early during the course of infecTABLE A V E R A G E OF H E M A T O L O G I C V A L U E S

>>

O

g X

R.B.C. million

7 IN

W.B.C. Hetnogl. (gram) thous.

15

Phase of Injection

Temp. F°

(Normal)

99.6

7.5

8.6

13.6

41

2 - 5 Days Foil. Inf.

100.7

6.8

6.9

12.8

1 st Fever Attack

105.8

5.8

6.0

Remissions

99.7

7.2

9.1

EXPERIMENTAL

Hema- Nentr tocrit

HORSES

Lymph. per

Mono. cent

Eos.

57

35

2

3

35

51

40

2

2

9.8

29

67

24

2

0

13.3

38

63

28

4

2

1 s.

At Fever Attacks

104.3

3.9

8.2

10.5

32

60

32

3

1

tò

Remissions

99.9

5.8

8.9

11.8

34

52

38

5

1

At Fever Attacks Remissions

102.6 100.6

5.1 6.9

9.8 9.0

11.3 12.6

34 36

58 45

30 41

3 8

2 2

vj -

3 0

I

tion, making its initial appearance in most cases during the first week, usually on the third to fifth day following an experimental infection. As a rule a decrease is clearly indicated before or during the first marked fever attack (Tables 4, 5, and 6 ) . At the onset of the fever response, or before, there is a shift in the relative proportion of the lymphocytes and the polymorphonuclear neutrophils: the number of the former diminishes considerably, thus producing a relative increase in the number of the neutrophil leukocytes (Tables 4 and 5 ) . During remissions between the first two or three fever attacks, the total number of white blood cells shows a tendency to return to the normal values; this is often accompanied by a transient relative lymphocytosis.

54

EQUINE INFECTIOUS A N E M I A

In m o r e advanced stages of the disease, the relative lymphocytosis remains a fairly constant feature of the blood picture. During relapses in subacute or chronic cases a reduction m a y b e observed in their relative n u m b e r , but this is not as p r o n o u n c e d as in the early, acute p h a s e of the infection. Some symptomless and well-balanced virus carriers show practically normal values in their differential white cell counts. O n the o t h e r h a n d , some chronic cases which, following very m u c h prolonged remissions, p r o duce relapses f r o m time to time may exhibit a fairly c o n s t a n t relative lymphocytosis. It is probably for this reason that, in the past, lymphocytosis was considered by some observers p a t h o g n o m i c of infectious anemia. There are descriptions in the literature, in which the correctness of infectious anemia diagnosis was criticized because of the low percentage of lymphocytes recorded in the condition. C a r e f u l observers, however, expressed the view that, simultaneously with the decrease of the red blood corpuscles, lymphopenia occurs during fever attacks ( 1 1 , 1 1 6 , 2 5 2 , 8 9 ) . O u r experience indicates that this is true for the first few fever attacks, but later the decrease of the lymphocytes during relapses is, in most instances, insignificant, especially following prolonged afebrile remissions. Long-standing chronic cases, manifesting clinical s y m p t o m s time and again, generally exhibit a relative lymphocytosis with little tendency to a decrease in spite of fever relapses. This may be illustrated by the h e m a tologic findings in one of our experimental horses. In this animal ( H o r s e 10), while it exhibited no clinical symptoms whatever f o r a b o u t six months, the average red cell count was 8.1 million, and the average of total white blood cell counts 9 , 7 0 0 in 18 bleedings p e r f o r m e d in the afebrile period. During the same time in the same bleedings, an average of 39 per cent lymphocyte was counted in the differential blood smears. This horse has had several fever relapses afterwards, f o r about five m o n t h s ; in this period the lowest erythrocyte and white blood cell counts r e c o r d e d were 6.6 million, and 6.300, respectively. However, the lymphocytosis r e m a i n e d unaffected during this time, as 12 differential counts gave an average of 36 per cent lymphocytes. T h e lymphocytosis, as seen in most chronic cases, is not quite stable, there is a fluctuation of the values u p and down. In our experimental animals the highest lymphocyte count was 6 0 per cent in the records; in general, values over 4 0 per cent were often noted in chronic cases. It must be a d d e d — a s already m e n t i o n e d — t h a t symptomless virus carriers may show normal hematologic values, indicating that an equilibrium of the infectious process is reached in such carriers. A definite decrease in the n u m b e r of eosinophils was regularly o b served during fever reactions particularly in the acute stage, but in most cases also during relapses of the subacute and chronic cases. A f t e r fever attacks their n u m b e r reached the normal level again. In H o r s e 10 a n o r m a l eosinophil count was revealed averaging 3 per cent during the six-month

7.

HEMATOLOGY

55

symptomless period mentioned above. W h e n fever relapses occurred later, the eosinophils d i s a p p e a r e d or decreased to 1 per cent several times. T h e n u m b e r of monocytes often increases during remissions following a few fever attacks ( T a b l e s 6 and 7 ) . I m m a t u r e , y o u n g f o r m s of the neutrophil granulocyte series are not recorded in the tables; usually neutrophilic metamyelocytes and staff cells, rarely myelocytes are sometimes seen in cases of prolonged duration and f r e q u e n t exacerbations. In o u r cases, as in most findings recorded in the literature, very little tendency was noted in the differential counts toward a shift to the left. Occasionally, leucocytosis was noted in the horses in a range of 11,500 to 15,800. N o correlation could be detected between these relatively rare values, m a k i n g a b o u t 5 per cent of all total white blood cell counts, and the s y m p t o m s of t h e disease. It is our impression that such higher values were m o r e c o m m o n in horses with a tendency to become chronic cases, and also in the ones receiving several injections in attempted immunization. W h e t h e r they indicate an increased reactivity of the hematopoietic system, or an easier mobilization of the white blood cells, is an undecided question. In brief, our findings indicate that, beside the fluctuating decrease and increase in the n u m b e r of the erythrocytes, a tendency to leukopenia and, later, a general trend to relative or absolute lymphocytosis can be noted during the disease process. However, it is difficult to set solid rules, as the blood picture is constantly changing. T h e findings in our horses concur, in general, with those obtained by most investigators reporting about the blood changes. T h e r e are, however, some differences between our findings and those recorded in the literature, especially with respect to the severity of the alterations, which we shall discuss later. *

It was early recognized that anemia is a regular symptom of the typical disease ( 2 5 6 ) , as may be seen f r o m the names used in various countries to designate the condition; it was considered characteristic of the disease by all of the early investigators. Lignée ( 2 5 6 ) , who first described infectious anemia as a clinical entity, referred to the condition as anhémie du cheval. Zschokke ( 4 9 8 ) and Ostertag ( 3 3 7 ) used the n a m e "pernicious anemia of horses," indicating that they assumed some analogy between the h u m a n disease a n d that of the horse. T o r r a n c e ( 4 5 6 ) observed m a r k e d anemia in cases of s w a m p fever in C a n a d a , but he emphasized that poikilocytosis was never seen in those horses. C a r r é a n d Vallée ( 5 8 ) reported that, in their experimental horses, the drop in the n u m b e r of the red blood cells preceded the fever reaction as the first sign of the disease. O n the 10th to 16th day following infection, they noted a decrease by one and one-half to two million against the n o r m a l n u m b e r of red blood cells, setting the latter at 7 million. In acute cases there was a f u r t h e r decrease until death of the animal ensued, while cases

56

EQUINE

INFECTIOUS

ANEMIA

of longer duration showed values as low as a b o u t one million per cubic millimeter. During remissions an increase was observed; this, however, did not reach the level of the original normal values. T h e y also recorded a slight decrease in the n u m b e r of white blood cells and r e m a r k e d that the lymph glands were always involved in the process, showing hyperemia and slight hemorrhages in acute cases and a m a r k e d swelling in the chronic p h a s e ; enlargement of the spleen was also present in most of their cases. Other investigators soon confirmed and extended the findings reported by C a r r e and Vallée. Studying equine anemia in the United States, M a c k (271 ) stated that the decrease in the n u m b e r of red blood cells o c c u r r e d gradually and reached low values similar to those recorded by the F r e n c h investigators; the total n u m b e r of the white blood cells remained in the normal range or decreased slightly. Kinsley ( 2 0 5 ) has presented a good description of the changes in the blood. H e studied not only the behavior of the red cells, but also the alterations in the differential c o u n t of the white blood cells. Van Es and associates ( 4 7 0 ) were struck by the regular presence of a marked anemia in field cases and its rather u n c o m m o n occurrence in cases in which the disease was experimentally p r o d u c e d . Seyderhelm ( 4 0 5 ) recorded observations similar to those of V a n E s and co-workers and expressed the view that the equine disease is closely related to pernicious anemia in man. Wirth ( 4 8 7 ) did not concur with this opinion, as he had not observed anisocytosis in equine infectious a n e m i a ; therefore, he considered it a severe type of anemia with n o relation to human pernicious anemia. Jaffé ( 1 8 8 ) concluded f r o m his careful histologic studies that the bone marrow is not primarily involved in the equine disease, but may show signs of reaction in very severe, prolonged cases; he f o u n d the main cause of anemia in an injury to the red blood cells and in their massive destruction during fever attacks. Seyderhelm ( 4 0 5 ) described the hematologic picture both in the circulating blood and in the organs. He found in the blood stream no indication of an increased hematopoiesis but observed, at the same time, markedly reddened b o n e m a r r o w along with myeloid changes in the secondary blood-forming organs; he noted all stages of erythropoiesis and leukopoiesis in the liver and the spleen of infected horses. His conclusion that the condition is related to h u m a n pernicious anemia was not accepted by others. Most authors agree now that tli ere is no direct relationship between the two diseases, a view expressed by Hutyra and Marek ( 1 8 0 ) as early as 1910. Dobberstein ( 8 4 ) related that in the equine disease no alterations can be noted in the shape of the red blood cells; he concluded f r o m this that the anemia is not caused by the primary action of the virus exerted on the hematopoiesis. For the unusual findings of some investigators (poikilocytosis, elevated color index, e t c . ) . two explanations may be offered. O n e of them is that the changes noted in a very much prolonged stage of the disease may differ considerably from the typical blood picture, most likely on account of the exhaustion of the blood-forming organs. T h e other possibility is that the

7.

HEMATOLOGY

57

controversial hematologic findings were noted in horses with mixed infection (page 2 4 ) ; this is frequently f o u n d in the temperate zone and is probably quite c o m m o n in tropical regions. N o r m o b l a s t s and other i m m a t u r e f o r m s of the erythrocyte series are seldom f o u n d in the blood even in prolonged cases. Liihrs ( 2 6 4 ) stated that in h u n d r e d s of differential counts m a d e on blood of infectious anemia horses, normoblasts were noted in a single instance only. W e saw nucleated red blood cells in two of our experimental horses; one of them ( H o r s e 4 ) had a red cell c o u n t of 2.5 million when this was recorded during the 9th prolonged fever attack. In earlier blood smears of the same horse n o normoblasts were noted. M a r e k ( 2 7 5 ) stated in 1921 that the volume of the packed cells ( h e m a t o c r i t ) , which a m o u n t s to f r o m 40 to 50 per cent in normal horses, drops considerably in the early stage of infectious anemia. A l t h o u g h statements have been m a d e to the contrary, it would seem that the hemoglobin decrease largely follows the decline in the n u m b e r of the red blood cells ( 4 9 3 , 1 3 4 ) . T h e resistance of the erythrocytes, as measured in solutions of different osmotic strength, was found to be generally increased ( 5 6 0 , 317, 2 6 1 ) . On the other hand, there is also observation on record that it decreases during the fever paroxysm ( 1 1 ) . L e u k o p e n i a and a decrease or disappearence of eosinophils during fever attacks was mentioned in the early reports ( 4 5 6 , 58, 2 0 5 ) . T h e r e was a divergence of opinion f o r a considerable time as to the relative participation in the decrease, or increase, by the lymphocytes and the p o l y m o r p h o n u c l e a r neutrophils, but the dispute may at present be considered settled ( 1 1 , 2 5 0 , 149, 1 1 6 ) . T h e findings vary according to the phase of infection, as briefly discussed on page 54. Variations are noted also in the reaction exhibited by individual animals. Similarly, the degree of anemia as noted in individual horses varies considerably. F r o m the normal figures the red cell count may progressively go below one million ( 5 8 , 11, 317, 2 5 0 ) . T h e r e are some differences according to countries and authors in what is considered normal value. Scarborough collected the data published by 23 authors on normal horses; the average f o u n d in 3 1 8 counts was 7.8 million R B C (red blood corp u s c l e s ) . It is known that the values in thoroughbred horses are considerably higher: according to a study recently published by Hansen and co-workers ( 1 5 3 ) in A m e r i c a , the average red cell count in 65 thoroughbred mares was f o u n d to be 10.5 million. In our horses the average normal count noted was 7.5 million R B C ( T a b l e 7 ) . T h e lowest figure that we recorded in our investigations was 1.4 million, and the average of the lowest values in 20 infected horses sufficiently well studied was 4 , 1 5 0 , 0 0 0 R B C . This indicates that the cases on which we are reporting may be considered less severe than the average field cases, probably because our experimental horses were not worked and were kept under o p t i m u m conditions.

58

EQUINE

INFECTIOUS

ANEMIA

Concerning the nature of anemia, it may be said that the disease cannot be classified as one of the more common anemias. According to a widely used classification ( 2 1 6 ) , the anemias form two large classes: 1) the anemias due to blood loss and destruction, characterized by hyperactivity of the bone marrow, and 2) anemias due to decreased blood production with hypoactivity of the bone marrow. A further classification is based on size and hemoglobin content of the red blood corpuscles: the common deficiency anemias are usually microcytic and hypochromic, while pernicious anemia is characterized by macrocytic, hyperchromic cells. In our infected experimental horses the erythrocytes appeared normal in size, shape, and staining qualities; accordingly, the anemia is normocytic, and normochromic in its general character. This is in line with the experience of the large majority of other investigators in this field. The anemia is chiefly due to an excessive destruction of the red blood cells ( 1 8 8 ) indicated by the marked hemosiderosis in the liver (see Fig. 9 on page 128). S;ill it does not completely fit into the first class as mentioned above. It cannot be listed among the so-called secondary anemias, neither can it be grouped with the hemolytic anemias, and it is not related in its pathogenesis to human pernicious anemia. With this negative characterization, equine infectious anemia must be placed in a separate subgroup of its own. There is a possibility that anemias of somewhat similar behavior reported to occur in sheep and goats (90, 662) and in cattle ( 8 7 ) might have a relationship to the equine disease. It is often difficult to differentiate infectious anemia from other types of anemia that are fairly common in some countries. In Sweden, Finland, and Switzerland nutritional anemia seems to occur side by side with genuine infectious anemia in certain regions. Huguenin ( 1 7 9 ) considered many of the anemia cases in Switzerland as a deficiency disease due to undernourishment. Krupski and associates (224, 226) believed that most of the equine anemias noted in Switzerland are of streptococcus origin; Keller ( 1 9 4 ) has shown by experiments that transmissible and nontransmissible anemias exist, while Steck (415, 418) maintains that equine infectious anemia is widespread in Switzerland. Svanberg ( 4 4 3 ) considers the soil and vegetation responsible for the noncontagious anemias in Sweden. Laurell ( 2 3 9 ) and Carlstrom and Hjarre (53) also stated that two types of anemia exist in that country, one of them being of nutritional origin. In the latter type the reticuloendothelial system is not involved ( 5 3 ) . The use of the hematologic findings for the diagnosis has been reviewed by several authors (122, 169, 170, 235, 252, 3 8 5 ) . According to Schermer and his co-workers (385, 387) the diagnosis may be ascertained in 70 per cent of the cases by following the changes in the blood regularly. A systematic study of the blood changes on repeated occasions may be a valuable aid in making a diagnosis, if combined with other laboratory tests and careful clinical observations.

CHAPTER 8

HEMAGGLUTINATION Hirst ( 1 6 4 ) discovered in 1941 that cultured influenza virus would cause agglutination of the erythrocytes of the chicken embryo in which the virus was cultivated. W h e n the test was performed with cells of normal chicks, the same p h e n o m e n o n occurred. The hemagglutination was considered a result of the specific action of the virus. This concept was supported by the fact that a specific influenza immune serum would inhibit the agglutination ( 1 6 5 ) . Since 1941, a number of h u m a n and animal viruses have been shown to p r o d u c e hemagglutination as seen in the Hirst test, e.g., the viruses of variola and vaccinia ( 5 2 ) , Newcastle disease ( 5 1 ) , mumps, etc. A n attempt was made to develop a similar hemagglutination test in equine infectious anemia. F o r this purpose one of us began a systematic study of the problem at the early phase of the present investigations during the summer of 1948, and reported the preliminary findings in a note published in 1949 (Dreguss ( 9 4 ) ) . Before discussing the results of the hemagglutination test and their significance in infectious anemia, an outline of the method will be given and the preliminary findings will be summarized. Methods and Preliminary Results.—To obtain the chicken red blood cells, normal chickens were bled aseptically by heart puncture or from the wing vein; about an equal volume of 3.8 per cent sodium citrate solution was used as anticoagulant. The red blood cells were washed three times with sterile physiologic salt solution in the centrifuge and a 10 per cent suspension of packed cells was prepared. This stock cell suspension, well stoppered, should be " a g e d " at room temperature for a few days before use; it will then remain usable for several weeks, if kept at refrigerator temperature under aseptic conditions to prevent contamination. Small test tubes, 10 mm. wide and 75 mm. long, with round-shaped bottoms are suitable for setting up the test. First, two-fold dilutions of the serum are m a d e ( 1 : 1 0 , 1:20, e t c . ) ; then to each 0.5 cc. of diluted serum is added an equal volume of 0.3 per cent suspension of the chicken red blood cells freshly prepared in physiologic salt solution from the stock suspension. Proper controls should be included, such as normal serum, a known positive serum, and a red blood cell suspension with no serum added. (59)

60

EQUINE INFECTIOUS

ANEMIA

In general, the t e c h n i q u e similar to t h a t used in h e m a g g l u t i n a t i o n tests with the viruses of influenza ( 1 6 5 ) , o r N e w c a s t l e disease ( 3 2 ) , w a s followed. R e a d i n g is d o n e a f t e r 6 0 to 7 5 m i n u t e s s t a n d i n g of the t u b e s u n d i s t u r b e d at r o o m t e m p e r a t u r e . If t h e test is negative, t h e s e d i m e n t e d cells f o r m a c o m p a c t , sharply d e m a r c a t e d disc at t h e b o t t o m of the t u b e s . P o s i tive h e m a g g l u t i n a t i o n is indicated by small c l u m p s of cells finely d i s p e r s e d t h r o u g h o u t the b o t t o m of the t u b e in a n u n e v e n e d g e d w i d e layer. R e s u l t s are expressed in t e r m s of the highest dilution of s e r u m o r o t h e r test m a t e r i a l which p r o d u c e s a definite h e m a g g l u t i n a t i o n . A s described in the p r e l i m i n a r y r e p o r t ( 9 4 ) , all 16 h o r s e s t h a t w e r e inoculated with the virus of infectious a n e m i a until t h e t i m e of t h e r e p o r t developed agglutinating s u b s t a n c e s in their blood f o r n o r m a l c h i c k e n red blood cells. Titers were r e c o r d e d u p to s e r u m dilutions of 1 : 3 2 0 d u r i n g the early stage of the illness, while they d e c r e a s e d s o m e w h a t d u r i n g the s u b a c u t e stage; a s y m p t o m l e s s c a r r i e r elicited low titers. F o r t h e p u r p o s e of control, a n u m b e r of n o r m a l h o r s e s w e r e tested. T h e i r sera e i t h e r did not p r o d u c e h e m a g g l u t i n a t i o n o r did so in dilutions of 1 : 1 0 to 1 : 2 0 only. T h e r e were indications t h a t positive results m a y be o b t a i n e d in c o n d i t i o n s o t h e r than infectious a n e m i a . It was a s s u m e d at the t i m e of the r e p o r t t h a t the h e m a g g l u t i n a t i o n is c a u s e d either ( a ) by t h e i n f e c t i o u s agent, ( b ) b y antibodies, o r ( c ) by s o m e p a t h o l o g i c c o m p o u n d p r o d u c e d d u r i n g t h e c o u r s e of the disease a n d present in the b l o o d of infected a n i m a l s . F r o m these preliminary findings it s e e m e d w o r t h w h i l e to p u r s u e f u r t h e r the investigations in t w o m a i n d i r e c t i o n s : first t o find o u t w h e t h e r the h e m a g g l u t i n a t i o n can be utilized as a m e a n s of diagnosis if a specific inhibition test can be d e v e l o p e d ; secondly, to clarify the m e c h a n i s m of t h e reaction for better u n d e r s t a n d i n g a n d f o r gaining insight into the i n f e c t i o u s process. T h e results of these efforts will be briefly p r e s e n t e d below.

Diagnostic

Application

of the Hemagglutination

Test

First it was a s s u m e d that the h e m a g g l u t i n a t i o n is a n a l o g o u s to t h a t seen with influenza and o t h e r viruses. T h i s a s s u m p t i o n h a d s o m e s u p p o r t in that the severity of the disease a p p e a r e d to be c o r r e l a t e d with t h e agglutination titer of the serum o r p l a s m a of the infected horses. In a f e w experimental horses that exhibited less p r o n o u n c e d s y m p t o m s t h a n the average, the agglutination titer not only d e v e l o p e d slowly b u t n e v e r r e a c h e d as high a level as in the m a j o r i t y of animals. H o r s e 2, which at first s h o w e d m a r k e d clinical s y m p t o m s a n d , at the s a m e time, e v i d e n c e d a high agglutinating titer in its blood serum ( u p to a dilution of 1 : 3 2 0 ) , b e c a m e a s y m p t o m l e s s virus carrier a f t e r a few attacks, a n d f r o m t h e n o n w a s in robust health, resisting re-infection with large doses of highly v i r u l e n t virus; coincident with this the original high titer d r o p p e d s h a r p l y , a n d soon r e a c h e d the level c o n s i d e r e d to be in the n o r m a l r a n g e . In o r d e r to d e m o n s t r a t e the specific n a t u r e of the h e m a g g l u t i n i n , inhibition tests were set u p with s e r u m of h o r s e s c o n s i d e r e d as b e i n g in

8.

HEMAGGLUTINATION

61

(he convalescent stage. In general, no specific inhibition was demonstrated by the use of such serum samples, or of any other type of serum or plasma collected in various stages of the disease. T h e results were contradictory and failed to show any regularity. If inhibition was noted in certain setups, normal serum seemed to produce a similar effect. It was realized after much unsuccessful effort that the system being tested was too complex. Although acute-stage serum samples were utilized as the source of hemagglutinin ( v i r u s ? ) , and later-stage serum samples as inhibitors, theoretically both could contain virus as well as antibody, since horses once infected usually remain lifelong carriers of the virus. M o r e over, a specific reaction, if it did occur, would probably be influenced by such side reactions as were seen in the precipitin and colloid stability tests when diluting or mixing normal and pathologic serum samples. W e were confronted with a situation different from the known inhibition tests in which immune sera are tested against cultured virus or, in some instances, clarified supernatants of organ suspensions containing the virus. M o s t of the inhibition tests in our experiments were performed according to the technique used in influenza work ( 1 6 5 ) or in Newcastle disease ( 3 2 ) . M a n y modifications of these methods were tried in an effort to overcome the difficulties and eliminate nonspecific reactions; because of the inconclusive results, the details are not given here. During our experimental studies the hemagglutination test itself was routinely applied to our horses and the changes were closely followed in all of them. T h e r e was not a single experimental horse in our series that did not show a rise of hemagglutination titer following infection by an active virus, whether it was a primary infection or a challenge inoculation. T h e r e fore, the conclusion seems to be justified that hemagglutination, using serum or plasma of infected horses and suitable chicken red blood cells, is a regular feature in typical infectious anemia. Thus, a characteristic, even if not quite specific, alteration in the blood of infected horses is strongly suggested. In T a b l e 8 we have summarized the highest titers observed in individual horses receiving primary inoculation of a virulent virus; the day when this titer was noted and the gamma-globulin level, determined simultaneously, are also recorded. Horses with marked fever attacks during the acute and subacute stage, usually soon developed a high hemagglutination titer that persisted for a longer time, lasting even during remissions of fever, although with a tendency slowly to decrease. In cases of a more chronic nature, the severity of repeated fever attacks was not as closely correlated with the hemagglutination titer of serum: some horses showed a definite rise, while others exhibited only minor changes in the titer during relapses. If we analyze the titers in horses subjected to experimental immunization, or utilized for testing cultured virus material and virus strains carried through mouse passages, the results are not as uniform as in inoculations with the typical active virus. Three horses ( H . 2 4 , H . 2 7 , and H . 2 8 )

62

E Q U I N E INFECTIOUS

ANEMIA

used in testing chick embryo material in attempted cultivation of the virus elicited an early mild elevation of the titer on the 5th to 8th day following inoculation; about a two- to three-fold rise was noted. A similar slight, early increase was noticed in a few instances following infection with h o r s e passage strains, if frequent bleedings were done. Such titers would decrease b e f o r e a sharp rise occurs later about at the time of the first m a r k e d fever attack. Such early mild reactions were also noted in horses that received material which had been subjected to mouse passages previously. E v e n in TABLE

8

HEMAGGLUTINATION T E S T IN E X P E R I M E N T A L L Y I N F E C T E D H O R S E S ( P r i m a r y Inoculation with Virulent Virus) Horse No. Horse Horse Horse Horse Horse Horse Horse Horse Horse Horse

3 4 7 8 16 17 20 22 26 29

Highest titer

Day foil, infection

Gammaglobulin

1:160 1:240 1:160 1:80 1:320 1:160 1:240 1:320 1:1280 1:320

29th 28th 32nd 63rd 17th 20th 27th 29th 10th 18th

2.43g 2.06 *

Note

*Not

done

1.96 2.32 2.27 2.98 2.52 2.21 2.35

the third serial passage ( H o r s e 3 8 ) , m a d e for the p u r p o s e of regaining the virulence of the virus to horses, only a very slight increase was r e c o r d e d in the hemagglutination titer of the serum accompanied by a m a r k e d , although delayed, fever attack. T h e impression was that the cultured virus and the mouse passage strains h a d lost the ability to p r o v o k e a m a r k e d hemagglutinin production. Horses inoculated with formalinized organ suspensions f o r i m m u n i z a tion, exhibited fairly high titers in their serum ( 1 :80 to 1 : 1 6 0 ) during the process of immunization, o n e of the horses in the early p h a s e ( H . 2 5 ) , another at the later stage of immunization ( H . 3 0 ) . A possible explanation is that the virus was attenuated but not completely inactivated in the vaccine used, thus causing a mild subclinical infection indicated by the positive hemagglutination test. Following challenge inoculation, the hemagglutination titers showed further increase in all immunized horses, as well as in those injected f o r testing the egg culture virus. T h e same was true for horses injected with mouse passage material, if and when they were subjected to challenge inoculation with horse passage virus. A s may be seen f r o m T a b l e 9, the peaks of the hemagglutination titers reached after the challenge inoculation

8.

HEMAGGLUTINATION

63

are comparable with those noted following primary inoculations. (For more details on these experimental horses, see Chapters 10, 11, and 12.) To obtain comparative values in horses other than the ones used in our experiments, a considerable number of normal blood samples were tested, in addition to those coming from field cases suspect for infectious anemia, or horses suffering from other diseases. Normal Blood.—Over 100 normal horse serum samples were tested, including those taken from the experimental horses before inoculation. TABLE

9

HEMAGGLUTINATION T E S T FOLLOWING CHALLENGE INOCULATION Horse No.

Highest titer

Day following inoculation

Gammaglobulin

Remarks

H.

5

1:640

20th

2.64g

monkey blood

H.

10

1:320

16th

2.28

mouse material

H.

15

1:320

14th

2.20

mouse material

H . 21

1:640

22nd

2.66

immun,

H. 24

1:160

15 th

1.89

egg culture

H . 25

1:240

21st

2.60

immun, (organ susp.)

H . 27

1:320

2 3 rd

1.94

egg culture

H . 28

1:320

22nd

2.00

egg cult, ( p a s s a g e )

H. 30

1:640

22nd

2.37

immun, (organ susp.)

H . 31

1:160

23rd

2.46

i m m u n , (organ susp.)

(alum)

The overwhelming majority of both groups either did not produce hemagglutination, or elicited a positive test in a 1:10 dilution only; some possessed a titer of 1:20; these groups comprised about 90 per cent of the normal horses. Thus, hemagglutination in a 1:20 dilution or less was considered as the normal range. The other 10 per cent of the clinically normal horses showed higher titers ( 1 : 4 0 to 1 : 8 0 ) . The question naturally arises, whether they were normal in every respect. Their past history was not known to us: they were usually old horses, and it is entirely possible that a recent illness or some chronic condition—not readily recognizable clinically—was responsible for the positive result. The higher titers observed in the blood of the horses were probably not due to a past infection by the anemia virus, since the disease was not prevalent in the environment from which these animals had come; but it is not quite impossible that some of them were infected previously. Owing to the limited facilities at our disposal, no horse inoculation test could be performed using the blood of these horses to rule out such a possibility, except in connection with crucial experiments. Horses with higher than normal titer were utilized for plain passage of the virus.

64

EQUINE

INFECTIOUS

ANEMIA

Blood of Sick Horses.—Many b l o o d s a m p l e s w e r e received t a k e n f r o m h o r s e s suspected of infectious a n e m i a m a i n l y b e c a u s e of u n e x p l a i n e d f e v e r attacks, w e a k n e s s , a n d a n e m i a . H e m a g g l u t i n a t i o n w a s o f t e n h e l p f u l to clarify the diagnosis, a l t h o u g h in s o m e of the field cases it w a s i m p o s s i b l e to follow the case history as, o f t e n , n o f u r t h e r i n f o r m a t i o n o r a d d i t i o n a l blood samples were available. F r o m regions w h e r e t h e disease is k n o w n to be prevalent (e.g. in the S o u t h ) , s e r u m s a m p l e s w e r e sent to us, c o m i n g f r o m h o r s e s diagnosed on the basis of clinical o b s e r v a t i o n s as h a v i n g infectious a n e m i a . T h e y o f t e n exhibited positive h e m a g g l u t i n a t i o n of similar titer as seen in h o r s e s infected e x p e r i m e n t a l l y . U n f o r t u n a t e l y , t h e s e l a b o r a t o r y results could not b e verified by h o r s e i n o c u l a t i o n tests; n e v e r theless, part of these investigations is interesting e n o u g h t o w a r r a n t a brief description. In 1950, we received 4 1 b l o o d s a m p l e s f r o m F l o r i d a w i t h o u t a n y p r e liminary i n f o r m a t i o n a b o u t the individual horses. T h e first 2 5 b l o o d samples c a m e f r o m o n e f a r m : in this g r o u p 2 0 w e r e positive, 4 q u e s t i o n able, a n d 1 negative by the h e m a g g l u t i n a t i o n test. I n f o r m a t i o n received f r o m the veterinarian in charge, a f t e r h e h a d l e a r n e d the results of t h e hemagglutination test, stated t h a t " t h e s e a n i m a l s a r e all very eligible f o r s w a m p fever. T h e h o r s e s are p a s t u r e a n i m a l s a n d are in p o o r c o n d i t i o n . M a n y of these tested animals s h o w severe a n e m i a a n d five of t h e h o r s e s have clinical s y m p t o m s of s w a m p f e v e r . " It m a y be a d d e d t h a t all of t h e five horses, identified by n a m e , exhibited strongly positive h e m a g g l u t i n a tion. It was also stated t h a t "six o r seven h o r s e s h a v e died f r o m clinical s w a m p f e v e r " d u r i n g the p r e c e d i n g y e a r o n the s a m e f a r m . T h e next 16 blood s a m p l e s c a m e f r o m a t h o r o u g h b r e d h o r s e f a r m at which t w o clinical cases of s w a m p f e v e r h a d been o b s e r v e d p r e v i o u s l y . F o u r of the samples were positive, a l t h o u g h the c o r r e s p o n d i n g a n i m a l s seemed to be in good health at the t i m e of testing. O n e h o r s e t h a t , at a n earlier date, h a d shown s y m p t o m s suggestive of i n f e c t i o u s a n e m i a — b u t was r e p o r t e d healthy at the time of the t e s t — g a v e a negative result. W h e n the same animal h a d an atypical t e m p e r a t u r e f l a r e - u p t h r e e m o n t h s later, blood was received again a n d , b e c a u s e it s h o w e d positive h e m a g g l u t i n a t i o n this time, 2 cc. of the s e r u m was injected into a test h o r s e . T h i s h o r s e p r o d u c e d mild fever reactions on the 18th a n d 2 5 t h d a y following i n o c u lation, but without significant a n e m i a ; thus, the result w a s i n c o n c l u s i v e . Challenge inoculation with a k n o w n virus strain o n the 3 8 t h d a y w a s f o l lowed by m a r k e d fever a t t a c k s a c c o m p a n i e d first by an i n c r e a s e of t h e red blood cell c o u n t to 9.1 million, a n d later by a n e m i a within a m o n t h ( 5 . 8 million R . B . C . ) . T h u s , in the F l o r i d a g r o u p , l a b o r a t o r y findings w e r e in a fairly g o o d a g r e e m e n t with the clinical o b s e r v a t i o n s indicating t h a t the h e m a g g l u t i n a t i o n test was of practical value in s u p p o r t i n g the diagnosis. Value of Negative Evidence.—As already m e n t i o n e d ( p . 2 3 ) r e p e a t e d tests were m a d e o n two h o r s e s suspected of h a v i n g infectious a n e m i a b u t p r o v e d finally to h a v e m a l i g n a n t t u m o r s . T h e clinical m a n i f e s t a t i o n s w e r e

8.

HEMAGGLUTINATION TABLE

65

10

H E M A G G L U T I N A T I O N IN V A R I O U S CLINICAL C O N D I T I O N S OF H O R S E S Hemagglutination Titer

Identification

Clinical and

Diagnosis Observations

T h . Br. 1

1:80

A n e m i c since o n e y e a r ; n o t d o i n g

Texas

1:320

Equine infectious anemia

H.

well

1:160

Intermittent fever, pale m u c o u s

W. N. C.

1:160

S u s p e c t of e q u i n e i n f e c t i o u s a n e m i a

H. 2086

1:160

Edema

T h . Br. 3

1:80

Lost

R. W.

1:80

Jaundice

H. 708

1:160

Epizootic

C. 2

1:320

Infectious

T h . Br. 4

1:160

T e m p e r a t u r e e l e v a t i o n s , e d e m a of legs

Th. Br. 4

1:20

( S a m e h o r s e as a b o v e , o n e y e a r l a t e r )

C. 4

1:80

Mare; aborted

N. J.

negative

Purpura

L. M d .

negative

E p i d e r m o i d c a n c e r of t h e

F.

negative

D o e s n o t use h i n d legs at t u r n i n g

negative

H e a l t h y a n i m a l ( c h e c k u p ) . W a s sick e a r l i e r

H.

3003

S.

Phil. V. Hosp.

1:10

of t h e a b d o m e n .

much

Chronic

weight

membranes

Anemia

recently

lymphangitis abortion

haemorrhagica

heart

stomach

condition

S. H .

negative

Past contact with infectious a n e m i a case

H.

702

negative

Lost much weight recently

H.

702

negative

Weakness, diarrhoea

H.

806

negative

Sick h o r s e . N o c l o s e r

negative

Brucellosis?

A . S. C .

1:10

Chronic

anemia

V. H .

negative

Anemia

following

C. 1

negative

Streptococcus

C. 3

1:10

Anemic mare.

H. 448

information

accident

anemia Aborted

H. 644

negative

Anemia following trauma

B.

S.

negative

A b o r t e d t w o w e e k s a g o . Salmonella

T h . Br. 2

1:20

aggl. p o s .

M i l d icterus. N o t d o i n g well

F. T.

negative

Lost m u c h weight

W.

negative

D i s s e m i n a t e d t u m o r of p a r e n c h . o r g a n s

G.

66

EQUINE INFECTIOUS

ANEMIA

similar to those of infectious anemia, and fever reactions were noted from time to time; the total protein and the albumin-globulin ratio were considerably lowered. The hemagglutination titer, however, was consistently negative in both animals and the immune-globulin level of the serum remained in the normal range. On the basis of the latter two laboratory tests, we considered the diagnosis as negative for infectious anemia, but the horses were kept on the suspect list on account of the clinical symptoms. The laboratory diagnosis was fully verified by post-mortem findings. The autopsy disclosed, in the first case, a large epidermoid cancer of the stomach extending to the abdominal cavity (post-mortem diagnosis by Dr. J. W. Mills of the Veterinary School, University of Pennsylvania); and disseminated tumor of the liver, lungs, and kidneys in the second case (information received from the veterinarian who observed the case clinically and performed the post-mortem investigation). In some other instances, where bacterial origin of secondary anemia was established by showing the causative organism or high titer of specific antibodies (Streptococcus-infections, S. abortus equi, etc.), it is worthy of note that the chicken red blood cell hemagglutination was either completely negative, or produced a titer of low range. Table 10 summarizes part of the results obtained with serum samples of horses suspected of infectious anemia or some other disease. Many of these samples were received on request, as it was intended to test blood samples of horses in different pathologic conditions in order to compare the results with those obtained in infectious anemia. Other samples were sent in for diagnostic purposes to confirm or exclude infectious anemia in febrile conditions. The clinical diagnosis as listed in the table must be regarded as tentative in most instances. According to the experimental results as outlined above, it would seem, as observed in the preliminary report ( 9 4 ) , that a negative outcome of the hemagglutination test with serum of horses showing febrile reaction and other symptoms is fairly good evidence against infectious anemia; also, virus carriers in the quiescent period would usually exhibit titers close lo the normal range. On the other hand, interpretation of a positive test requires more caution. Combined together with other tests, it may be of use in confirmation of the clinical diagnosis, but it might well indicate also a pathologic condition other than infectious anemia possessing a similar infectious mcchanism. The test described in 1949 (Dreguss ( 9 4 ) ) has since been utilized by other investigators and the positive results have been confirmed by the use of scrum from experimentally infected horses tested against different individual chicken cells. (Personal communication by Dr. C. D. Stein, 1951.) Mention has been made of the fact that the hemagglutination test, like many other laboratory procedures suggested for supporting the diagnosis, is not specific for equine infectious anemia ( 3 9 0 ) , which of course was never claimed.

8.

Mechanism

67

HEMAGGLUTINATION

and Significance

of the

Hemagglutination

Efforts were made to clarify the mechanism of the hemagglutination test as observed with chicken red blood cells and infectious anemia serum of horses, with the hope that understanding of the hemagglutination may eventually throw light on the infectious mechanism of the disease. It would be beyond the scope of the present report to describe in detail the long series TABLE HEMAGGLUTINATION

Red

Blood Corp. of Horse

Horse 32 (5th d.)

Horse 32 (5th d.)

Horse 33 (5th d.)

!!

11

OF R E D B L O O D C O R P U S C L E S OF T H E

Blood Serum Tested Horse Horse Horse Horse Horse Horse *H. H. H. H. H. H. H. H.

17 10 10 32 32 32

856 884 1042 1251 1389 1389 1389 1389

Horse Horse Horse Horse

17 17 33 33

Taken Days Foil. Injection

HORSE

Hemagglut. Titer

(before) 13 70 (before) 17 20

1:40 1:640 1:2560 1:10 1: 1280 1:640

(carrier) (carrier) 28 13 (before) 13 15 23

1:80 1:160 1:320 1: 160 negative 1:40 1:80 1:320

(before) 20 19 25

1:10 1:160 1:640 1:1280

S e r u m s a m p l e s H . 856 to H . 1389 were received from the H . A . I . . W a s h i n g t o n .

of experiments which were performed for this purpose. Instead, a brief summary of the more important investigations will be presented. Hemagglutination of Horse Blood Cells.—Attempts were made in the early phase of the investigations to produce hemagglutination with red blood cells of n o r m a l and infected horses, but no hemagglutination was noted in the range of serum dilutions comparable to that seen with chicken cells. Sometimes, agglutination occurred in lower dilutions ( 1 : 2 , 1:5) attributable to iso-agglutinins of blood groups. In a few instances spontaneous agglutination was observed after collecting blood from infected horses; the horse blood cells evidenced a definite clumping together in their own plasma

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while s t a n d i n g at r o o m t e m p e r a t u r e o r in t h e r e f r i g e r a t o r . S u c h cells, a f t e r t h e y h a d b e e n w a s h e d t h r e e times in p h y s i o l o g i c salt s o l u t i o n , w o u l d e i t h e r t e n d to a g g l u t i n a t e even w h e n diluted w i t h saline, o r w o u l d lose t h e i r a g g l u t i n a t i n a b i l i t y f o l l o w i n g w a s h i n g . T h e results w e r e n o t b e t t e r w h e n t h e cells w e r e " a g e d " in t h e l a b o r a t o r y f o r a f e w d a y s , as w a s d o n e w i t h t h e c h i c k e n cells. By c o n s i d e r a b l y p r o l o n g i n g t h e p e r i o d a l l o w e d f o r this, s o m e g o o d results w e r e r e c o r d e d later. T h r e e h o r s e s w e r e injected f o r c h a l l e n g e i n o c u l a t i o n , e a c h w i t h a f e w cc. of virulent s e r u m , a n d five d a y s later b l o o d w a s d r a w n f r o m all t h r e e a n i m a l s , with the a s s u m p t i o n t h a t t h e b l o o d cells w o u l d h a v e h a d c o n t a c t with the virus by this time. T h e red b l o o d cells w e r e w a s h e d a n d utilized f o r the test as usual, b u t they g a v e c o m p l e t e l y n e g a t i v e r e s u l t w i t h i n f e c t i o u s h o r s e sera in a series of r e p e a t e d h e m a g g l u t i n a t i o n tests. A 10 p e r c e n t s t o c k s u s p e n s i o n of the w a s h e d cells w a s p r e s e r v e d at 5 ° C . a n d t h e tests w e r e r e p e a t e d a f t e r t w o m o n t h s , this t i m e with positive result. T h e results w e r e i m p r o v e d a f t e r t h e p r e s e r v e d cell s u s p e n s i o n w a s w a s h e d in salt s o l u t i o n a n d the test w a s set u p with 0 . 4 p e r c e n t cell s u s p e n s i o n i n s t e a d of t h e u s u a l 0 . 3 p e r cent. T h e b l o o d cells w e r e f r e e of b a c t e r i a l c o n t a m i n a t i o n a n d g a v e c l e a r - c u t results, p a r t i c u l a r l y t h o s e w h i c h c a m e f r o m t w o of t h e h o r s e s exhibiting a mild f e v e r r e s p o n s e on t h e t h i r d d a y f o l l o w i n g i n f e c t i o n , a n d several m a r k e d f e v e r a t t a c k s t h e r e a f t e r . T a b l e 11 illustrates s o m e of t h e "results n o t e d b y t h e u s e of t w o h o r s e cell s u s p e n s i o n s ( t h o s e of H o r s e s 3 2 a n d 3 3 ) . A s m a y b e n o t e d , t h e r e d b l o o d cells of b o t h h o r s e s w e r e a g g l u t i n a t e d b y t h e i r o w n s e r a c o l l e c t e d a f t e r i n f e c t i o n . N o r m a l sera s h o w e d c o n s i d e r a b l y l o w e r a g g l u t i n a t i o n titers. T h e s c c o n d g r o u p of sera o r i g i n a t e d f r o m h o r s e s e x p e r i m e n t a l l y i n f e c t e d with the virus of i n f e c t i o u s a n e m i a a n d k i n d l y f u r n i s h e d b y D r . Stein a n d D r . M o h l e r of t h e B u r e a u of A n i m a l I n d u s t r y in W a s h i n g t o n . T h e r e s u l t s with t h e s a m p l e s of H . 1 3 8 9 illustrate t h e d e v e l o p m e n t of h e m a g g l u t i n i n s f r o m the p r e i n o c u l a t i o n t i m e to t h e 2 3 r d d a y f o l l o w i n g i n f e c t i o n . T h e first two of t h e W a s h i n g t o n s e r u m s a m p l e s c a m e f r o m l o n g - t i m e v i r u s c a r r i e r s ; these e v i d e n c e d m o d e r a t e h e m a g g l u t i n i n titers, as seen in t h e T a b l e . T h e results w e r e readily r e p r o d u c i b l e . Hemagglutination with Various Red Blood Cells.—As reported earlier ( 9 4 ) . red b l o o d cells of s h e e p , g u i n e a - p i g s , a n d h a m s t e r s w e r e also t e s t e d f o r their suitability to t h e h e m a g g l u t i n a t i o n test; t h e y o c c a s i o n a l l y g a v e a positive result in tests with i n f e c t i o u s h o r s e s e r u m , b u t g e n e r a l l y t h e r e s u l t s were i n f e r i o r to those n o t e d with c h i c k e n cells. R a b b i t cells p r o d u c e d conflicting results a n d o f t e n m a n i f e s t e d s p o n t a n e o u s a g g l u t i n a t i o n . R e d blood cells of p r e i n o c u l a t e d r a b b i t s , i n j e c t e d with n o r m a l o r i n f e c t i o u s s e r u m of horses, elicited s t r o n g h e m a g g l u t i n a t i o n with a n y h o r s e s e r u m as an indication of a serologic reaction to the f o r e i g n p r o t e i n . E x p e r i m e n t s w e r e c o n d u c t e d on a large s c a l e f o r closer s t u d y of t h e agglutination of c h i c k e n red b l o o d cells. A d s o r p t i o n of t h e h e m a g g l u t i n i n and its clution w a s effected at v a r i o u s t e m p e r a t u r e s . T h e r e d cells t h u s treated w e r e f u r t h e r s t u d i e d in a d d i t i o n a l e x p e r i m e n t s d e v i s e d m a i n l y f o r

8.

HEMAGGLUTINATION

69

c o n c e n t r a t i n g the agglutinin; a m o d e r a t e c o n c e n t r a t i o n was achieved in these e x p e r i m e n t s , a n d s o m e p r o p e r t i e s of the h e m a g g l u t i n i n w e r e n o t e d . L a c k of s p a c e p r e c l u d e s a m o r e detailed description of these e x p e r i m e n t s , m o s t of t h e m of t h e o r e t i c a l significance; they w e r e originally designed f o r c o n c e n t r a t i n g the virus of infectious a n e m i a , in case it w e r e a c c o m p a n i e d by, o r w e r e identical with, the s u b s t a n c e causing agglutination of the erythrocytes. Serum Protein Fraction Containing the Hemagglutinin.—Chemical d e t e r m i n a t i o n s , c o m b i n e d with h e m a g g l u t i n a t i o n tests, disclosed that the h e m a g g l u t i n i n is c o n t a i n e d in the i m m u n e - e l o b u l i n f r a c t i o n of the s e r u m p r o t e i n s in i n f e c t i o u s h o r s e blood ( p . 7 6 ) . T h i s finding raised the q u e s t i o n , w h e t h e r the s u b s t a n c e responsible f o r the h e m a g g l u t i n a t i o n r e p r e sents the virus itself, or is r a t h e r an a n t i b o d y of peculiar n a t u r e . T h e latter possibility is s u p p o r t e d by m a n y analogies f o u n d in the m o d e r n literature d e a l i n g with the p r o b l e m of h e m a g g l u t i n a t i o n in various infectious diseases; beside, it fits into the c o n c e p t of the pathogenesis of infectious a n e m i a ( 9 5 ) d e v e l o p e d on the basis of o u r e x p e r i m e n t a l studies, as discussed in C h a p t e r 9.

In the literature of infectious a n e m i a , there are a few r e p o r t s related to h e m a g g l u t i n a t i o n . Several investigators, a m o n g t h e m Balozet ( I I ) , o b s e r v e d a u t o - a g g l u t i n a t i o n ( s p o n t a n e o u s a g g l u t i n a t i o n ) on s o m e s a m p l e s of infectious a n e m i a b l o o d . W e n o t e d the same p h e n o m e n o n several times, p a r ticularly w h e n b l e e d i n g h o r s e s in the early phase of a s t r o n g fever a t t a c k . O p p e r m a n n a n d L a u t e r b a c h ( 3 3 3 , 3 3 4 ) , on the o t h e r h a n d , tried to apply the h e m a g g l u t i n a t i o n f o r diagnostic p u r p o s e in utilizing red blood cells of r a b b i t s w h i c h h a d been previously injected with infectious h o r s e s e r u m ; they a t t r i b u t e d d i a g n o s t i c value to the positive results r e c o r d e d in the test. T h e p r o c e d u r e did not p r o v e useful in the h a n d s of re-investigators ( 2 8 , 2 2 2 , 2 9 6 ) , a p p a r e n t l y b e c a u s e of the serologic reaction to the foreign protein which d o m i n a t e s the test, as m e n t i o n e d a b o v e . B e f o r e a t t e m p t i n g to explain the possible m e c h a n i s m of the h e m a g g l u t i n a t i o n test with c h i c k e n red blood cells a n d with those of horses, we shall list several analogies which m a y c o n t r i b u t e to an u n d e r s t a n d i n g of the phenomenon: 1 ) Several viruses are k n o w n to p r o d u c e hemagglutination of chicken cells as m e n t i o n e d in the i n t r o d u c t i o n ( p . 5 9 ) . Hirst ( 1 6 4 ) , w h o first d e s c r i b e d s u c h h e m a g g l u t i n a t i o n in influenza, has shown that it is specifically inhibited b y the c o r r e s p o n d i n g i m m u n e s e r u m ( 1 6 5 ) . T h e s a m e is true f o r N e w c a s t l e disease virus ( 3 2 , 5 1 ) , a n d several o t h e r viruses. M o o l t e n a n d C l a r k ( 3 0 4 , 3 0 5 , 3 0 6 ) recently a t t e m p t e d to use the h e m a g g l u t i n a t i o n test in c l a r i f y i n g the infectious m e c h a n i s m of hemolytic a n e m i a a n d o t h e r b l o o d - b o r n e h u m a n diseases suspected to h a v e virus origin; they a t t r i b u t e an i m p o r t a n t role to a u t o - h e m a g g l u t i n a t i o n in these diseases. In our case, it could n o t be p r o v e n by inhibition tests that the hemagglutinin is identical with the virus ( p . 6 0 ) .

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2 ) By the very nature of the hemagglutination, the analogy with blood group iso-agglutinins is fairly close. T h e r e is, however, a difference in the strength of the reaction, which is manifested by the high titers of infectious anemia hemagglutinin during the active stage of the disease, and by its independence f r o m the blood groups of the individual horses. 3 ) In several h u m a n diseases hemagglutinins have been detected which are thought to represent a particular group of antibodies. T h e s e are the heterophile antibodies in serum sickness ( 7 4 ) , in infectious mononucleosis ( 3 4 5 , 1 3 0 ) , and in leukemia ( 5 5 ) ; the cold agglutinins in p r i m a r y atypical pneumonia ( 3 4 8 , 1 7 3 ) ; and the heterogenetic antibodies described in acute hepatitis ( 1 0 2 ) , etc. In the case of the heterophile agglutination with sheep red blood cells, the heterophile antibodies do not indicate an etiologic or other direct relationship to the red cells which they agglutinate. T h e cold agglutinins belong to the group of auto-hemagglutinins. T h e division between the agglutinin types in this group is not sharp. 4 ) There are hemagglutination tests known, in which an in vitro reaction of the antibody is brought about with red blood cells previously sensitized by various substances (e.g., with specific p o l y s a c c h a r i d e s ) , or by adsorption of viruses (usually Newcastle virus) to their surface. T h e sensitizing substance seems to modify the surface of the erythrocytes used in these tests, and thus prepares them for the hemagglutination reaction with the antibody to be tested. Such tests may be considered relatively specific, as in the case of the M i d d l e b r o o k - D u b o s test ( 2 8 8 ) f o r tuberculosis; or unspecific. as in the tests used for experimental studies in infectious mononucleosis ( 1 0 9 , 1 1 2 ) . and in rheumatoid arthritis ( 1 5 9 ) . T h e type and behavior of the hemagglutination noted in infectious anemia seems to be nearest to that mentioned in the groups under 3 ) and 4 ) . It is probable that the hemagglutinin present in the serum of infectious anemia horses is an antibody of heterogenetic nature. T h e hemagglutination test with chicken red blood cells may be readily explained by this. T h e details and the supporting evidence for this assumption will be presented in a separate publication ( 9 5 ) . T h e antigenic material that gives rise to the hctcrogenctic antibodies is, in all likelihood, derived f r o m the b r e a k - d o w n products of horse erythrocytes. Therefore, the hemagglutinin possesses a specific affinity to the red blood cells and is probably responsible f o r their excessive destruction in the horse. This concept is supported by the results noted in hemagglutination with long-stored horse blood cells ( T a b l e 1 1 ) ; it seems very likely that a partial (enzymatic) break-down m a d e these cells suitable for hemagglutination by infectious horse sera. T h e problem will be further discussed in the next chapter when the pathologic process as a whole is considered.

CHAPTER

9

PATHOGENESIS E q u i n e infectious anemia is perhaps one of the least understood infectious diseases. It has been known, since the f u n d a m e n t a l experiments by Vallée and C a r r é , that the disease is caused by a filterable virus but the closer mechanism of the infection has not been clarified, in spite of the great interest centered a r o u n d the malady and the many efforts to reach a better understanding of the disease and to gain a deeper insight into its infectious process. Although some features of the disease have been clarified by careful observations and experimental studies, there remain a n u m b e r of important questions unanswered which have been summarily called "the problem of infectious a n e m i a . " T h e disease is unquestionably infectious, but it does not behave as do most infectious diseases. With rare exceptions, no immunity follows a past infection in animals apparently recovered; such horses may experience relapses at any time, and their clinically healthy state can change to an acute fatal disease for no apparent reason. J a f ï é ( 1 8 8 ) concluded, after a careful analysis of the histologic changes, that the K u p f f e r cells of the liver take up an extremely large n u m b e r of crushed red blood cells that have been previously destroyed for an unexplained reason. Schermer ( 3 8 2 , 3 8 3 ) stated that the virus is insufficient to p r o d u c e the disease and to explain the pathologic process. Both he and Dobberstein ( 8 4 ) remarked that the histologic findings in infectious anemia are reminiscent of those produced by break-down products of a foreign protein and that the reticulo-endothelial system and the capillary walls are seriously involved in the process. M a n y others analyzed the question of pathogenesis ( 1 3 9 , 184, 297, 318, 3 8 6 ) to explain part of the changes, but the main p r o b l e m — t h a t is, the real cause of the alterations and the way they are i n d u c e d — r e m a i n e d unsolved. Systematic investigations, as part of our study, disclosed the fact that hypersensitivity plays an important role in the pathologic process of the disease t p d that the allergic state is responsible for many of the paradoxical findings: Hypersensitivity of shorter or longer duration can be observed in a n u m b e r of other infectious and non-infectious diseases as shown by von Pirquet ( 3 5 2 - 3 5 6 ) who coined the word "allergy" in 1906. T h e monograph on h u m a n serum sickness published by von Pirquet and Schick ( 3 5 6 ) in Vienna is the first systematic study on hypersensitivity, with theoretical deductions still valid five decades later. In a broader sense the term "allergy," (71)

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or allergic state, means altered reactivity of the organism; the more restricted meaning of allergy is a delayed reaction that follows the action (inoculation) of a sensitizing antigen (infectious or non-infectious in n a t u r e ) , while anaphylaxis ( 3 5 7 ) is an immediate response to such antigen in a sensitized organism. T h e term "hypersensitivity" can be applied to both conditions. T h e allergic sensitivity causes the sensitized organism to react in a m o r e intensified manner, different f r o m the reaction expected f r o m an i m m u n e organism. Allergy and immunity, although they are frequently present together, are not considered to be interdependent conditions ( 2 7 6 ) . In the following, the evidence indicating the role of allergy in the pathogenesis of infectious anemia will be briefly outlined. A m o r e detailed description will be published elsewhere ( 9 5 ) . Early Fever Reaction

and Marked

Fever

Attacks

The incubation period most frequently observed in infectious anemia moves in a range f r o m one to three weeks, the average being a b o u t 14 days, based on the occurrence of the first marked fever reaction and other symptoms. In most of our horses we noted that a mild t e m p e r a t u r e response, between the 2nd and the 6th day, preceded the sharp rise in t e m p e r a t u r e , which as a rule followed the first elevation 8 to 9 days later. T h e r e was a striking regularity in this interim period between the two t e m p e r a t u r e rises. Chart 1 illustrates the fever response in a young horse ( H o r s e 17) that h a d been injected with 1 cc. of a virulent infectious anemia serum collected f r o m Horse 10. T h e disease took an acute course in Horse 17 and the animal died on the 28th day following infection. F u r t h e r analysis of all t e m p e r a t u r e charts showed that in the early phase of the disease the t e m p e r a t u r e peaks often occurrcd after remissions of 7 to 10 days. We are convinced that the early fever reaction, that may be termed the "virus p e a k , " is the first sign of the action of the virus within the body of the infcctcd horse. It is safe to assume that a blood-borne virus does not require two to three weeks for multiplication in the original host; r a t h e r it would rcach within a few days sufficient concentration to cause noticeable symptoms, even if mild in character. Coincident with this, there is a decrease in the n u m b e r of the red blood cells (p. 4 9 ) . We were able to show by horse inoculation the presence of virus in the blood at this time. On account of the regular time interval between the fever reactions, and because of a papular eruption noted on an infected horse at the time of the second fever attack, the allergic nature of these manifestations was c o n sidered. It is known that in serum sickness and other allergic conditions the incubation period, as a rule, is from 8 to 10 days, sometimes longer. T h e fever response in human and experimental measles ( 9 2 ) seems to have a similar pattern; as it is known, von Pirquet ( 3 5 5 ) considered the skin eruption in measles as an allergic manifestation. T h e early mild fever reaction, noticed also by others ( 1 9 3 , 278, 2 8 0 ) , was not noted in some of our horses in spite of other systemic reactions present.

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It seems that the virus causes in the first days following the infection a slight initial reaction with or without a mild fever response. Then stronger symptoms and marked fever attacks follow, presenting the pattern of typical allergic fever reactions. Small laboratory animals injected with the virus seem to develop only the early mild reaction, if any. Challenge

Inoculation

and the Possibility

of

Re-injection

The existence of an allergic state in infectious anemia is strongly supported by the fact that all of our experimental horses reacted to challenge inoculation with minute amounts of virulent virus, even if there was plenty of evidence that they had passed a milder infection previously; often, the response following the second infection was stronger than that following the primary inoculation. T o bring additional proof, Horse 10 was injected with 0.5 cc. of its own blood serum, collected at the first marked fever reaction and preserved frozen in a sterile vial until it was used. This animal had been in a quiescent, afebrile period showing no fever reaction or other clinical symptoms for over five months when it was re-inoculated with its own serum, to which it responded with a somewhat accelerated fever reaction with a peak of 103.5° F. This finding leaves no doubt that horses seemingly recovered from an infection may be re-infected with the virus, the only exception being the resistant virus carriers which are apparently sufficiently desensitized. Observations concerning re-infection, or increased susceptibility are noted also in the literature (p. 1 2 3 ) . Antibody

Level

in the Blood and Activity

of the

Disease

According to our present knowledge the allergic symptoms are based on antigen-antibody reactions which are believed to take place on the surface of the cells within the sensitive tissues ( 4 6 3 ) . Therefore, the serum proteins in the blood of our infected horses were subjected to an intensive study. Attention was focused on the gamma-globulin fraction that contains most of the antibodies in infectious diseases ( 1 0 3 ) . The gamma-globulin has been determined routinely by the chemical method of Kibrick and Blonstein ( 2 0 3 ) , using 15 per cent sodium sulfate concentration for salting out this fraction of the serum proteins. In addition, a few preliminary determinations were performed by electrophoresis (81 ). In Tabic 12 are recorded the highest gamma-globulin values that we noted in the individual horses after infection. The total serum protein values given in grams for 100 cc. serum, and the erythrocyte counts recorded on the same day are also included. Normal values, as determined in the same horses before inoculation and in a number of other healthy horses, moved in a range from 1.17 to 1.68 grams of gamma-globulin per 100 cc. serum. As may be seen from the table, an unusual increase was noted in practically all horses. It is noteworthy that Horse 8 had a low value. This horse never showed marked clinical symptoms during the several months of observation following infection. In a resistant virus carrier (Horse 2 ) , the level of gamma-globulin, which reached very high values during the acute symptoms,

9.

75

PATHOGENESIS

returned close to the normal range, when the animal showed a complete clinical recovery. T h e peak of the gamma-globulin level was reached in all horses while they were in the active stage of the disease. Generally, an increased antibody level in the serum is considered as an indication of a past infection a n d — a t the same t i m e — a s a sign of relative or solid immunity. If the rise of the gamma-globulin level in infectious TABLE SERUM GAMMA-GLOBULIN

Horse No.

Highest y-globulin

LEVEL

Day Foil. Inf.

12

IN T H E B L O O D O F I N F E C T E D

R.B.C. million

Total Protein

H.

4

2 . 9 6 g.

94th

3.8

7 . 8 4 g.

H.

5

3.29

27th

4.8

8.39

H.

8

1.96

63rd

H.

11

3.60

80th

4.3

6.56

H.

15

3.33

36th

5.9

9.0

H.

21

3.61

78th

6.2

8.45

HORSES

Remarks

5.90

H.

22

3.57

59th

4.5

8.64

H.

27

2.14

30th

6.9

8.61

H.

28

2.82

24th

7.4

9.36

H.

30

2.57

33rd

5.7

8.77

H.

31

3.21

74th

9.8

7.38

H.

32

2.45

25th

6.5

7.03

H.

33

3.28

24th

5.8

8.05

H.

36

1.84

22nd

7.6

7.10

Mouse

pass,

H.

38

2.75

17th

6.7

9.30

Mouse

scries,

H.

40

2.96

35th

10.0

8.91

Cult, series,

Several chall.

H.

44

2.49

23rd

5.5

8.16

Mouse

H.

46

2.81

17th

8.2

8.25

Shock

virus 3rd 2nd

series. foil.

inoc.

3rd

inoc.

N o t done.

anemia represents an increase in antibodies, as it does in other infectious diseases, one would expect an amelioration or ceasing of symptoms coincident with the rise. But, as the case of Horse 2 clearly shows, recovery and a p p a r e n t immunity occur rather parallel with the decrease of g a m m a globulin a p p r o a c h i n g the normal level. This strongly suggests that the excess a m o u n t of antibody present in the circulating blood of infected horses is an indication of their allergic state, that is, of an increased readiness for antigenantibody reactions, recognized as characteristic for allergy. In recent years, m a r k e d hyperglobulinemia, similar to the one noted by us, was reported in various h u m a n diseases such as lupus erythematosus ( 7 1 , 4 7 3 ) , leukemia ( 3 8 ) , rheumatic arthritis ( 4 7 ) , lymphogranuloma venereum ( 4 7 7 ) , and liver diseases ( 1 2 5 ) , with extremely high values in hepatic necrosis ( 4 9 7 ) . I n all of these, the increase was attributed to ele-

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INFECTIOUS

ANEMIA

vated gamma-globulin level as determined by chemical methods or by electrophoresis; in two of the reports ( 3 8 , 125) the subfraction was m o r e closely identified as g a m m a - o n e globulin (also called beta-two f r a c t i o n ) . V a u g h a n and co-workers ( 4 7 3 ) noted correlation between the gamma-globulin content of serum and the duration of the disease in lupus erythematosus. It may be added that Longcope and R a c k e m a n n ( 2 6 0 ) d e m o n s t r a t e d the relation of circulating antibodies to manifestations of serum sickness. Antibodies, considered as modified serum globulins ( M u d d ( 3 1 4 ) ) , are mostly contained in the regular gamma-globulin (i.e., in the g a m m a two globulin). T h e g a m m a - o n e fraction corresponds to a protein which has about six times as high molecular weight as the regular antibodies; its molecular weight (in the horse s e r u m ) is 9 9 0 , 0 0 0 as against 150,000 of the normal gamma-globulin ( 4 6 1 ) . According to Tiselius and K a b a t ( 4 4 9 ) pneumococcus antibodies, produced in horses by h y p e r i m m u n i z a t i o n , migrate with the g a m m a - o n e fraction in the electrophoretic field. It seems from our preliminary electrophoresis analyses that the excessive a m o u n t of antibody present in infectious anemia serum belongs to the same g r o u p of heavy globulins and its mobility is that of the g a m m a - o n e globulin. V a n der Schecr and co-workers ( 4 0 8 ) studied the antibody in h y p e r i m m u n e horse sera prepared against 15 different bacterial antigens; they f o u n d that some of these antibodies were contained in the regular y-globulin fraction ( g a m m a - t w o ) , while others migrated with the " T " c o m p o n e n t . We made a few analyses by electrophoresis with a Tiselius type of apparatus at the D e p a r t m e n t of Medical Physics, University of Pennsylvania.* This confirmed the unusual increase in the gamma-globulin fractions of the serum proteins in sick horses. By c o m p a r i n g the electrophoresis pattern of normal horse serum ( C h a r t 2 ) with that recorded on serum of an infected animal ( C h a r t 3 ) , an enormous increase may be noted in the so-called gamma-one fraction which is located between the beta- and gamma-globulin fraction and is considered by most investigators as a g a m m a globulin variety; it is also called " T " component, or beta-two globulin ( 4 6 1 ) . The albumin boundary (A ) indicates a m a r k e d d r o p in that c o m p o n e n t . The sera of horses indicating a d r o p in the g a m m a - g l o b u l i n — a s d e t e r m i n e d by the chemical m e t h o d — p r e s e n t e d an electrophoresis pattern of considerably diminished g a m m a - o n e globulin fraction. The Role

of Hemagglutinin

in the Pathologic

Process

As already mentioned in C h a p t e r 8, the c o m p o n e n t responsible f o r the hemagglutination was found to be present or to be associated with the gamma-globulin fraction of infectious anemia serum. By salting out this fraction and dissolving it in saline, the hemagglutinin was almost q u a n t i t a tively separated f r o m the rest of the serum protein fractions. * T h e electrophoresis analyses were performed with permission of Dr. Britton C h a n c e , head of the Department, in collaboration with Dr. A n d r e a s C. M a e h l y , and Dr. Gerhard F r o m m .

9.

77

PATHOGENESIS

T h e g a m m a - g l o b u l i n f r a c t i o n of infectious sera p r o b a b l y contains several types of a n t i b o d i e s ( t o the virus, and to the " s e c o n d a r y " a n t i g e n s ) . A m o n g these the h e m a g g l u t i n i n seems to possess c o n s i d e r a b l e significance as to the role it might play in the pathologic process. It causes agglutination in

A

CHART

2. ELECTROPHORESIS

PATTERN

OF N O R M A L

HORSE

SERUM

vitro with b o t h c h i c k e n red blood cells and h o r s e e r y t h r o c y t e s . It is most likely that the s a m e h e m a g g l u t i n i n will p r o d u c e h e m a g g l u t i n a t i o n in vivo, t h a t is, in t h e c i r c u l a t i n g blood of the infected horse, w h e n p r e s e n t in the s e r u m in sufficient c o n c e n t r a t i o n . Bull showed in 1 9 1 5 the o c c u r r e n c e of

CHART

3.

ELECTROPHORESIS

PATTERN

OF

INFECTIOUS

ANEMIA

SERUM

i n t r a v a s c u l a r h e m a g g l u t i n a t i o n by bacterial antibodies, a n d Kniscly and cow o r k e r s convincingly d e m o n s t r a t e d such agglutination ( s l u d g e d b l o o d ) in several p a t h o l o g i c p r o c e s s e s of living animals ( 2 0 7 , 3 3 ) . B l a n c h a r d and V e d e l ( 4 0 ) r e p o r t e d t h r o m b o s i s in the h e p a t i c ( p o r t a l ) and splenic veins in i n f e c t i o u s a n e m i a . W e o b s e r v e d in histologic p r e p a r a t i o n s of the spleen of an i n f e c t e d h o r s e , d e s t r o y e d a f t e r p r o l o n g e d f e v e r reaction, a m a r k e d agglutination of t h e red b l o o d cells. All this points to the direction that the excessive d e s t r u c t i o n of the red blood cells is at least partly d u e to the

78

EQUINE

INFECTIOUS

ANEMIA

hemagglutinin present in the s e r u m ; the virus p r o b a b l y acts in sensitizing the red cells a n d b r e a k i n g d o w n s o m e c o m p o n e n t of the cell s t r o m a . A t high t e m p e r a t u r e s ( f e v e r ) the hemagglutinin m a y be eluted f r o m the red b l o o d cells, which b e c o m e r e f r a c t o r y to r e p e a t e d h e m a g g l u t i n a t i o n . It is a s s u m e d that the antigen which gives rise to the p r o d u c t i o n of h e m a g g l u t i n i n s in infectious a n e m i a is a p o l y s a c c h a r i d e of h a p t e n n a t u r e , p r o b a b l y derived f r o m the s t r o m a of the red blood cells of the infected horse. This is based, in p a r t , on analogies of e x p e r i m e n t a l w o r k r e p o r t e d in the literature ( 5 0 , 3 9 3 , 3 9 4 , 4 5 3 , 4 5 4 ) . T o m c s i k a n d his c o - w o r k e r ( 4 5 4 , 3 9 3 , 3 9 4 ) studying similar p r o b l e m s in c o n n e c t i o n with s e r u m sickness a n d the h e t e r o p h i l e agglutination in infectious m o n o n u c l e o s i s , h a v e s h o w n in their e x p e r i m e n t a l studies the role of h a p t e n s . Microscopic

Evidence

of Allergic

Inflammation

A l t h o u g h n o histologic alterations exist which w o u l d specifically indicate the presence of an allergic state, there are c h a r a c t e r i s t i c .changes which regularly o c c u r in allergic i n f l a m m a t i o n s . T h e s e h a v e been o b s e r v e d in infectious a n e m i a . H e r e , a brief r e f e r e n c e will be m a d e to a few of t h e m , while the details will be discussed later ( C h a p t e r 1 3 ) . In o u r e x p e r i m e n t a l h o r s e s infiltration, mostly perivascular, by l e u k o cytes a n d l y m p h o c y t e s in various o r g a n s , including the h e a r t , h a s been n o t e d with regularity. In the m y o c a r d i u m the perivascular infiltration ( F i g . 1 ) is followed by proliferation of c o n n e c t i v e tissue elements, o f t e n resulting in lesions strongly resembling Aschoff bodies ( 9 ) considered to be specific f o r r h e u m a t i c h e a r t fever (see Fig. 15 on p. 1 3 1 ) . Eosinophil cells infiltrate in large n u m b e r the liver ( F i g . 2 ) , the lymph n o d e s (Fig. 3 ) , a n d the spleen ( F i g . 1 2 ) . T h e increase of p l a s m a cells ( F i g . 4 ) indicates a n t i b o d y p r o d u c t i o n , as often seen in h y p e r i m m u n i z e d animals as well as in allergic i n f l a m m a t i o n . T h e kidney changes, as shown in Figs. 5 a n d 6, are similar to

EXPLANATION

OF COLOR

(opposite

PLATE

page)

FIG. 1. HEART. P e r i v a s c u l a r i n f i l t r a t i o n in t h e m y o c a r d i u m . L y m p h o c y t e s a n d p o l y m o r p h o n u c l e a r l e u k o c y t e s a r e s u r r o u n d i n g a n a r t e r i o l e . ( H o r s e 9, H e m a t o x y l i n Eosin. approx.iOOx) FIG. 2. LIVER. M a r k e d i n f i l t r a t i o n by r o u n d cells a n d b y n u m e r o u s e o s i n o p h i l cells. T h e liver cells s h o w signs of r e g r e s s i o n . ( H o r s e 38, G i e m s a , a p p r o x . 5 6 5 x ) FIG. 3. LYMPH NODE. M o d e r a t e i n f i l t r a t i o n by e o s i n o p h i l cells. ( H o r s e 9, M e t h y l e n bltie-Fosin. a p p r o x . 5 0 0 x ) FIG. 4. H i ivvrir LYMPH NODE. T h e m a r g i n a l s i n u s o i d s a r e p a c k e d w i t h p l a s m a cells. ( H o r s e 10. H e m a t o x y l i n - E o s i n . a p p r o x . I 2 0 0 x ) FIG. 5. KIDNEY. P e r i v a s c u l a r i n f i l t r a t i o n by r o u n d cells in t h e c o r t e x of t h e k i d n e y . Signs of fibroblastic a c t i v i t y m a y be n o t e d . ( H o r s e 9, M e t h y l e n b l u e - E o s i n , a p p r o x . 5 0 0 x ) FIG. 6. KIDNEY; s e g m e n t of a g l o m e r u l u s . P r o l i f e r a t i o n of t h e c a p i l l a r y e n d o t h e l i u m , o b l i t e r a t i n g t h e B o w m a n s p a c e . ( H o r s e 21, H e m a t o x y l i n - E o s i n , a p p r o x . 5 0 0 x ) Micropho'ographs

by Dr. Steven

C.

Mohos

Plate: E q u i n e I n f e c t i o n s A n e m i a '

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FiG. 1 1 . Cellular infiltration and endothelial cell proliferation in the liver. Endothelial cells, plasma cells, lymphocytes, and polymorphonuclear neutrophils in the sinusoids. (Horse 1, Hemat.-eos., 125x.)

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FiG. 1 2 . Infiltration of eosinophils and other inflammatory cells in the spleen. In upper right corner part of a trabecula. (H. 21, Hematoxylineosin, 475x.) Microphotographs

by Dr. S. C.

Mohos

tive changes of the hepatic cells, proliferation of bile ducts, hemosiderosis, and lymphocytic infiltration. Hydropic degeneration and fatty metamorphosis involve the liver cells not altered by necrosis. Fatty degeneration is more pronounced around areas of necrosis. The necrotic process appears to have no predilection for any one portion of the liver lobule. Approximately one out of every three lobules has undergone partial necrosis. Only in reparative changes does this phase vary from the acute. Regeneration of liver cells is most apparent at the periphery of the lobules. Newly formed liver cells stain deeply acidophilic and their nuclei are hyperchromatic. Frequently groups of newly formed liver cells take the form

130

E Q U I N E INFECTIOUS A N E M I A

of acini or tubules that are not easily distinguishable from newly formed bile duct tubules (Fig. 10). Hemosiderosis is marked. Kupffer cells are heavily laden with the pigment, and free macrophages are enlarged by the engulfed hemosiderin. The free hemosiderocytes are-found in the central veins, sinusoids, periportal spaces, and in necrotic areas. There is a noticeable increase in the number of Kupffer and endothelial cells lining the liver sinusoids.

FIG. 1 3 . C e l l u l a r i n f i l t r a t i o n of t h e e n d o c a r d i u m by polymorphonuclear n e u t r o p h i l s a n d l y m p h o c y t e s . ( H . 7, Hematoxylin-eosin, 475x.)

FLG. 1 4 . P e r i v a s c u l a r i n f i l t r a t i o n of the a d r e n a l c a p s u l e . A s m a l l vessel is s u r r o u n d e d b y l y m p h o c y t e s . ( H . 3, Hematoxylin-eosin, 240x.) Microphotographs by Dr. J. W, Mills

The lymphocytic infiltration is intense. Lymphocytes, both mature and immature forms, are commonly seen between the endothelium of the sinusoids and the hepatic cords, in areas of necrosis, in the connective tissue of the portal triads and in the perilobular connective tissue, as well as packing blood vessels and sinusoids. Together with the hemosiderin laden macrophages, the lymphocytes frequently replace areas in which hepatic cells have been completely obliterated. With Wilder's reticulum stain it can be seen that there is an increase in the recticulum fibrils around the central vein and sinusoids of the majority of the liver lobules. The splenic changes consist of a marked reduction in hemosiderin content and in the number of erythrocytes as compared with the findings in the acute phase. Common lymph node alterations are: small focal hemorrhages and hemosiderosis. The pigment is partly extracellular and partly engulfed by

13.

Histopathology

131

macrophages. Proliferation of endothelial cells as well as a few inflammatory cell types are frequently seen (Fig. 19). The argentophil structure remains essentially unaltered (Fig. 20). The following renal changes exist in the majority of cases classified as subacute: infiltrations of lymphocytes and plasma cells are prominent around Malpighian corpuscles and around blood vessels. The glomeruli are more cellular and frequently fill Bowman's space completely. There

F l G . 1 5 . V a s c u l a r lesion in t h e h e a r t . P r o l i f e r a t i o n of c o n n e c t i v e tissue surr o u n d i n g a n arteriole in t h e m y o c a r dium and infiltration of m o n o n u c l e a r and m u l t i n u c l e a r cells. ( H . 1, H e m a toxylin-eosin, 3 0 0 x . )

F l G . 1 6 . C a r d i a c histiocytes ( " A n i t s c h k o w " cells) in t h e heart in longitudinal a n d cross section. A higher magnification of an area adjoining Fig. 15. ( H . 1, Hematoxvlin-eosin, 475x.) Microphotographs

by Dr. J. W.

Mills

are cellular adhesions between the glomerular loops and Bowman's capsule. The capillary loops are almost bloodless. The argentophil framework of the loops is more prominent and Bowman's capsule is thickened. There is a light deposition of hemosiderin pigment in the reticulo-endothelial cells and occasionally in the parenchymal cells of the convoluted tubules. Regressive changes in the tubules are most prominent in the convoluted ones. Tubular cells are seen in various stages of necrosis and many are desquamated. In the medulla there is interstitial edema, lymphocytic and plasma cell infiltrations, as well as scattered foci of necrosis. The intima and adventitia of the renal arterioles are edematous. There is fibroblastic proliferation in the adventitial coat. The arterioles and capillaries of the adrenal are surrounded by lymphocytes and plasma cells (Fig. 14).

132

EQUINE INFECTIOUS ANEMIA

Consistent cardiac findings are: myocardial muscle degeneration and necrosis, fibrinoid degeneration of connective tissue, cell aggregates of lymphocytes, Anitschkow or Aschoff cells, neutrophils and eosinophils. Hyaline degeneration and necrosis of cardiac muscle fibers occur as scattered focal lesions. Fibrinoid degeneration involves the interstitial and perivascular connective tissue (Fig. 1 5 ) . The existence of fibrinoid degeneration is determined by its metachromatic staining ability, as well as its positive reaction to Schiff's and to the periodic acid leukofuchsin reagents. Accumulations of lymphocytes, Aschoff cells, single and multi-nucleated, neutrophils and eosinophils are frequently seen in association with areas of perivascular fibrinoid degeneration and thus resemble the Aschoff body pathognomonic for rheumatic fever (Fig. 1 5 ) . The above cell types are also found interstitially, but apparently not in association with blood vessels (Fig. 16). Not all the above cell types are present in each lesion, however; the lymphocytes and the Aschoff cell are by far the most constant. The cardiac blood vessels most often altered are the arterioles. The changes in order of frequency are: infiltration of lymphocytes and Aschoff cells, edema of the adventitia and media, intimal hyalinization and fragmentation of the internal elastic lamina. The cardiac lesions resembling those of rheumatic fever will be recorded in greater detail in a separate publication ( 9 9 ) . The endothelial cells of the pulmonary arterioles and capillaries contain hemosiderin. The lumens of these vessels are partially or completely filled with macrophages containing hemosiderin. Microscopic

Alterations

in the Chronic

Stage

Chronic with Recurrent Fever Attacks.—Animals grouped under this classification and destroyed during or shortly after a fever attack exhibited hepatic lesions identical in character, though less extensive, than are noted in the subacute phase. Horses killed at a period from 15 to 20 days after the last fever reaction did not exhibit the same hepatic lesions; that is, necrosis was absent and either little or no indication of regenerative changes remained. In those animals destroyed during or shortly after a fever reaction, the alterations are: degenerative, necrotic and regenerative changes of the hepatic cells, proliferation of bile ducts, hemosiderosis and infiltration of lymphocytes and plasma cells. The normal liver pattern has been extensively altered in that the liver parenchyma has been replaced by fat vacuoles, mature and immature lymphocytes, plasma cells, and hemosiderin laden macrophages. In an occasional case numerous erythrocytes are seen replacing the necrotic parenchyma.

13.

HISTOPATHOLOGY

133

Chronic cases that were killed 15 to 20 days after their last fever reaction exhibited a marked lymphocytic infiltration and a definite hemosiderosis. In addition varying degrees of hepatic parenchymal regeneration and bile duct proliferation are occasionally seen (Fig. 10). The splenic changes are: a noticeable reduction in hemosiderin content and lymphoid hypoplasia. The lymph follicles are small and reduced in number. Eosinophils and other inflammatory cells are occasionally seen

FlG. 1 7 . Periglomerular and interstitial round cell infiltration and proliferation of endothelium of the capillary loops. Adhesions between the capillary tufts and Bowman's capsule. (H. 21, Hematoxylin-eosin, 125x.)

F l G . 18. A distended lymphatic vessel in the kidney containing various cells. Between the tubules are lymphocytes and fibrocytes. Tubular changes are minimal. (H. 21, Hematoxylineosin, 565x.) Microphotographs by Dr. S. C. Mohos

(Fig. 12). Consistent lymph node changes are proliferation of reticuloendothelial cells, hemosiderosis, the presence of numerous plasma cells, and multinucleated giant cells (Figs. 4, 21, 22). The germinal centers are enlarged. Hemosiderin pigment is partly extracellular and intracellular within macrophages. Numerous macrophages contain a phagocytized material not as yet identified. The multinucleated giant cell has a homogeneous, acidophilic staining cytoplasm, with from two to eight vesicular nuclei located at ths periphery of the cell (Fig. 22).

134

EQUINE INFECTIOUS A N E M I A

Common renal findings are: lymphocytic infiltration, increased cellularity of glomerular loops, thickening of Bowman's capsule, degenerative changes in the cells of the convoluted tubules, hyaline tubular casts, and hemosiderosis (Figs. 5, 6, 18). Foci of lymphocytes are observed near Malpighian corpuscles and blood vessels. Occasionally such foci resemble lymph follicles, having a light staining center that contains immature forms of lymphocytes (Figs. 5, 6, 17).

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F l G . 2 0 . A r g e n t o p h i l s t r u c t u r e of a lymph follicle. T h e light a r e a represents t h e sinusoids. T h e s t r u c t u r e appears well p r e s e r v e d . ( H . 9, Silver impregn., 5 6 5 x . ) Microphotographs

by Dr. S. C.

Mohos

Cardiac pathologic changes are comparable to those of subacute cases, with the exception that myocardial necrosis was absent in all the hearts studied (Fig. 1). Myocardial degeneration, in the form of hyaline degeneration, fibrinoid collagen degeneration, and aggregates of lymphocytes and Aschoff cells are occasionally observed. Valvular alterations were similar to those observed in the acute phase. Chronic Balanced or Asymptomatic Carrier.—The liver alterations are: fatty metamorphosis, lymphocytic infiltration, hemosiderosis, fibrosis, and bile duct proliferation. The fatty changes are concentrated in the periphery of the lobules. Lymphocytic infiltration is predominantly periportal and perilobular. Hemosiderin is present in small quantities in free and

13.

HISTOPATHOLOGY

135

fixed macrophages throughout all lobules. Wilder's reticulum stain demonstrates an increase in reticulum about the central vein and sinusoids of the liver. Increased amounts of collagenous connective tissue are noted periportally and perilobularly. T h e perilobular bile ducts are increased in number. Splenic changes consist of lymphoid hypoplasia and sparse hemosiderin. T h e lymph node architecture is partially obscured by mature and im-

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F l G . 2 2 . G i a n t cell in a h e p a t i c l y m p h node. A large multinucleated giant cell in t h e s a m e l y m p h n o d e a s in F i g . 2 1 . ( H . 11, H e m a t . - e o s . , 4 7 5 x . )

F l G . 2 1 . P l a s m a cells in a h e p a t i c l y m p h n o d e . M a n y p l a s m a cclls, b o t h mature and immature, are present. ( H . 11, H e m a t o x y l i n - e o s i n , 4 7 5 x . )

Microphotographs by Dr. J. W. Mills

mature forms of lymphocytes and plasma cells, and numerous macrophages containing hemosiderin, fat vacuoles, and other unidentified material (Fig. 4). T h e main renal alteration is a lymphocytic and plasma cell infiltration that appears in scattered foci throughout the kidney. In the lungs, macrophages laden with hemosiderin and lymphocytes fill the alveolar capillaries and larger pulmonary vessels in whole or in part. *

Certain gross pathologic alterations have been associated with equine infectious anemia f r o m its earliest recognition. The general body changes of emaciation, anemia, edema and jaundice were noted by those first describing the malady in 1843 (68, 78, 2 5 6 ) . Petechial hemorrhages involving many organs, splenic enlargement, alterations in the size, color and

136

EQUINE INFECTIOUS A N E M I A

consistency of the liver, and lymph node enlargement were noted by Carré and Vallée (56, 57, 5 8 ) . Similar gross findings have been reported from various parts of the world by numerous investigators studying the disease experimentally produced or in proven field cases. The gross pathologic lesions characteristic of the disease have been treated by Van Es and associates ( 4 7 0 ) in the United States; Todd and Wolbach ( 4 5 1 ) in Canada; the Japanese Commission ( 1 9 1 ) in Japan; deKock ( 2 1 1 ) in South Africa; Schermer ( 3 8 2 ) in Germany; Verge ( 4 7 6 ) in France; Dobberstein ( 8 4 ) and Fortner ( 1 1 5 ) in Germany; Gindin ( 1 3 9 ) in Russia; Stein ( 4 2 6 ) in the United States; Balozet ( 2 8 ) in North Africa; Cassamagnaghi and Cassamagnaghi ( 6 2 ) in South America. The microscopic findings described by Mack ( 2 7 1 ) in 1909 were extravasation and degeneration of erythrocytes in the major organs, leucocytic exudation in the vicinity of the vascular walls of the major organs, parenchymatous degeneration and necrosis of the liver, heart and kidneys, pigmentation of the liver and lymph nodes. These are basically the sam~ as those described later by Finzi ( 1 1 1 ) in France; Todd and Wolbach ( 4 5 1 ) in Canada; Jaffé ( 1 8 8 ) in Austria; Mòcsy ( 2 9 4 ) in Hungary; deKock ( 2 1 1 ) in South Africa; Schermer ( 3 8 2 ) in Germany; Hjârre ( 1 6 8 ) in Sweden; Balozet ( 2 8 ) in North Africa; Gindin ( 1 3 9 ) and Fedorov ( 1 1 0 ) in Russia; Stein ( 4 2 6 ) in the United States; and Epstein ( 1 0 7 ) in South America. Special studies have been made on the typical histologic alterations in individual organs. The changes in the lungs were studied in detail by Steck and Hauser ( 4 2 3 ) ; the heart by Dobberstein and Wilmes ( 8 8 ) , Matthiessen and Glasser ( 2 8 3 ) , and Schaaf, Hecke and Bert ( 3 7 9 ) ; the kidney by Leinati ( 2 4 8 ) ; the central nervous system by N a k a m u r a and associates ( 3 1 9 ) ; the spleen by Gindin ( 1 3 8 ) ; the liver by Hjârre ( 1 6 8 ) ; the lymph nodes by Piening ( 3 4 9 ) ; and the bone marrow by Wright ( 4 9 2 ) . Diagnosis based upon the histologic picture has been considered by Ziegler ( 4 9 6 ) and Nòller and Dobberstein ( 3 2 2 ) . While gross and microscopic findings may be cumulatively helpful in making a tentative diagnosis ( 4 2 6 ) , none is pathognomonic for the disease, nor are all the typical changes taken together sufficient for a positive diagnosis. Several systems have been considered as the primary site of viral attack. Most authors are in accord that the red blood cell is primarily affected, with rapid destruction of erythrocytes and subsequent accumulation of break-down products in the spleen. Early in the disease process the spleen becomes overloaded and its function of handling blood pigments exhausted, and consequently the burden is placed on the reticuloendothelial systems of other organs, such as the liver and lymph nodes. Another site of injury is the capillary endothelium, as indicated early in the disease by the common occurrence and general distribution of petechial hemorrhages. The cause of the increased capillary permeability has been attributed to the direct action of the virus ( 4 9 6 ) , to inflammatory

13.

HISTOPATHOLOGY

137

changes in the capillary walls, or to injury by toxic break-down products ( 1 3 8 ) . In response to the injury of the reticulo-endothelial cells and to the increased burden of pigment phagocytosis, the system undergoes proliferation. The bone marrow has been considered as a site of viral action; however, hyperplasia of the red marrow occurs in any disease after marked destruction of erythrocytes, provided that its power of regeneration is not destroyed. The bone marrow changes, though studied in only a few of our experimental animals, were striking only when the anemia was marked. In such cases, erythrocytic hyperplasia was apparent. The nature of the anemia as seen f r o m examinations of the peripheral blood as well as organ changes is that of a normochromic, normocytic anemia and does not simulate the alterations found in pernicious anemia of man. The degenerative and necrotic changes that occur in the parenchymal cells of the major organs may be the direct result of viral injury ( 2 8 ) , or a result of break-down products of the host tissue primarily altered by the virus ( 1 3 8 ) . The significance of the cardiac lesions resembling Aschoif bodies, the eosiniophilic cell infiltrations of the liver, spleen, and lymph glands, and the renal alterations as possible allergic manifestations will be considered in a separate publication ( 9 9 ) . In concluding the analysis of the microscopic alterations noted in the organs of our experimental animals infected with the virus of infectious anemia, it may be said that the changes show various phases of an inflammatory process, acute, subacute, or chronic in nature. The alterations reflect, on one hand, the direct systemic response of the infected animals to the invading virus, and, on the other hand, a histologic response indicative of the possibility that an allergic component may be present in the infectious process, particularly in the subacute and chronic phase of the disease, as discussed under the heading of Pathogenesis (Chapter 9 ) . The injury caused by the virus initiates, as described above, degenerative and necrotic processes around the vascular system of the parenchymatous organs, chiefly the liver. In a later stage of the disease process, infiltration of various inflammatory cells, mainly those of the lymphocytic series, is a prominent feature. In the parenchymatous organs, reparative proliferation of the tissues takes place with an increase of connective tissue elements that may lead finally to scar formations. As an example of such proliferation, the Aschoff body-like formations may be mentioned which were observed in the heart. The involvement of the reticulo-endothelial system is marked in cases of longer duration, exhibiting rich proliferation of the cellular elements of the reticulo-endothelium; this reflects an intensified reaction on the part of the defense mechanism in the infected animal. Noting this, particularly

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in prolonged chronic cases, some investigators suggested the name of "reticulo-endotheliosis" for use instead of that of infectious anemia. Hemosiderin deposits in the organs are the histologic expression of injury to a large number of red blood corpuscles. Hemosiderosis manifests itself most strikingly in the liver and, to a lesser degree, in the spleen and the lymph nodes. Anemia, i.e., a more or less marked decrease in the number of the red blood cells, although it is part of the pathologic process and has a considerable clinical significance, is by no means the only important feature of equine infectious anemia. It is rather the inflammatory process in its various phases that characterizes the disease, although neither the gross, nor the microscopic alterations can be considered as specific for the condition.

CHAPTER

THERAPY

AND

14

CONTROL

Therapy.—For the treatment of equine infectious anemia the cures suggested have been numerous; this is inevitable in the case of an infection that does not respond to any therapy. First many kinds of supporting or symptomatic therapy were tried; then chemotherapeutic treatment was attempted by several investigators. But it seems that all efTorts have failed, so far, to find an effective therapy and, in particular, to destroy the virus within the body of the sick animals or virus carriers. Sometimes, one cure or another seemed successful in suppressing symptoms, but in most cases this was very likely due simply to the spontaneous remission that is part of the typical disease. At the beginning of the chemotherapeutic era, Atoxyl and Trypanblue were among the first drugs to be used by research workers studying infectious anemia. Atoxyl was applied by Francis and Marsteller (123) in 1908, as well as by Dietrich ( 8 3 ) in 1910. The curative effect of Trypanblue was tested by Francis and Marsteller ( 1 2 3 ) and by Whitehouse (485) in 1911. The results following the use of these two drugs were questionable or negative. Neosalvarsan was also tried for treatment apparently without success ( 5 2 5 ) . The therapy used in nutritional or various types of secondary anemia in humans was also tried by many investigators. Larsson (237) administered iron, calcium, and phosphorus compounds, but the results were not promising. Gerlach ( 1 3 2 ) concluded that no therapy was ever successful in his hands. The use of homologous blood in the form of transfusions as well as normal horse serum was reported to give encouraging results. Rozsa ( 3 7 7 ) noted definite improvement in two-third of a group of infectious anemia horses by giving them transfusions from healthy donors, but one-third of the group did not respond to this treatment. Duvaux ( 1 0 1 ) found that administration of normal horse serum was quite helpful. Lamarre ( 2 3 3 ) reported that autotransfusions brought about improvement within 2 to 8 days, while Bailer (524) observed that autohemotherapy was only partly successful. The favorable results may probably be attributed to the stimulating effect of homologous blood on hematopoiesis ( 5 4 ) . (139)

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The use of nonspecific protein therapy in veterinary medicine was advocated by Fortner ( 1 1 4 ) , but Metzger ( 2 8 6 ) attempted the treatment of infectious anemia with parenterally administered protein preparations unsuccessfully. Mocsy ( 2 9 9 ) has seen no curative effect from large doses of normal bovine serum; the same was true for treatment with iodine (Lugol-solution). Schermer ( 3 8 3 ) in 1929 suggested the use of treatment similar to the one effective in human pernicious anemia. Following such line in the therapy, Krupski ( 2 2 5 ) fed infected horses with fresh liver of horses, calves, swine, and cattle in amounts varying from 8 to 26 kilograms. Some favorable results were noted, but they were not lasting. Verge ( 4 7 6 ) mentioned the parenteral use of Whipple liver extracts, but this procedure did not bring quite satisfactory results either. Among the numerous pharmaceutic preparations that have been utilized in the treatment of the disease are arsenical compounds, quinine, mercurial preparations, bismuth, antimony, various acids, vitamins, and enzymes. A long list of drugs and chemicals was used in a series of experiments to estimate their therapeutic value by Stein, Osteen, and Mott ( 4 3 7 ) in 1941. None of the drugs or chemicals tested were found to be effective. The sulfanilamides were also tested as to their value in the therapy by Gochenour and co-workers ( 1 4 1 ) , but were found to have no specific action on the malady or the virus causing it. Mott, Stein, and Heishman ( 3 1 1 ) reported in 1946 that penicillin had little or no effect on the course of the disease. While aureomycin has been used successfully in the treatment of some cases of human pernicious anemia by Lichtman, Ginsberg, and Watson ( 2 5 5 ) , and in the acute phase of viral hepatitis by Shaffer and co-workers ( 4 0 7 ) , its use in the treatment of equine infectious anemia has not been reported. In conclusion it may be said that, at present, we know of no therapeutic agent that has a curative action in equine infectious anemia. Supportive therapy by the use of blood transfusions, homologous serum, and parenteral administration of liver and iron deserve further trial, sincc several authors have reported favorable or suggestive results following their therapeutic use. W e believe that hypersensitivity is responsible, to a great extent, for the severe symptoms; therefore, any treatment that might lower the allergic reactivity in the horse will probably have its place in the symptomatic therapy of the disease even though it cannot ultimately cure the malady by acting on the virus itself. Control.—The problem of controlling the spread of infectious anemia is as old as the recognition that the disease is infectious. Effective control faces a number of obstacles; the weakest point of control measures is the difficulty of detecting the symptomless virus carriers. Since successful

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control depends upon the identification and eradication of infected horses, no systematic program can be undertaken until a specific, practical, and inexpensive means of diagnosis of all infected animals is developed ( 4 2 6 ) . The asymptomatic carrier horse probably constitutes one of the most common sources of virus in nature, but the reliable method of detecting such carriers, the horse inoculation test, is costly and time-consuming. For preventing the spread of infection, various control measures have been suggested. These are designed not only to spare the individual horse owner considerable losses, but also to protect the horse population within a community and this is often of great economic importance ( 3 9 6 ) . Practical measures to serve such a purpose are: 1) reporting all horses suspected of having the disease or carrying the infection, 2) separating them from the ones not affected by the disease, 3) killing either all suspected horses, or at least the proven cases, 4 ) branding suspected and proven cases if, for some reason, they cannot be killed. It is quite obvious that destroying all proven cases of the infection is, in the final analysis, less expensive than keeping such animals, thus preserving the source of contagion. It has also been proposed to designate some particular regions where chronic cases and carriers can be used for work ( 3 7 1 ) , if the disease is enzootic and widely spread in that area, but unrestricted shipping and export of horses from such regions should be forbidden. Control measures have been studied and discussed in detail by a number of authors in various countries (36,1 17,157,172,191,202,275,281, 396,426,476,499). In some countries official government regulations have been adopted for enforcing necessary control measures. Among these are Germany (particularly Prussia), France, Switzerland, Norway, Denmark, Italy, and Slovakia in Europe; Japan in Asia. References related to some of these control regulations can be found under the name of the country in our bibliography. In Germany, official laboratory tests for aiding the diagnosis are performed by central laboratories (668,1 17,122,729). The Office International des Epizooties in Paris ( 3 2 4 ) proposed measures that could be adopted on an international basis. Recently Fliickiger ( 1 1 3 ) discussed the efforts made in this direction and summarized the suggestions I'or international collaboration in the matter. Degen ( 7 7 ) , studying the problem of official government control measures in Switzerland, estimated that it cost the government 123,130 Swiss francs in Baselland canton alone to take the necessary steps for control during the years of 1930 to 1942. For 306 horses that were destroyed (average value estimated 1000 francs per horse) part-compensation was paid to the owner by the government. Over 29,000 francs of the total government cost mentioned above were used for control measures: investigations, therapeutic trials, autopsies, disinfection of stables. Thus, the average cost of these control measures amounted to 96 francs per horse, while the rest of the sum was spent for compensation.

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It is impractical to enumerate all the regulations and suggestions proposed for control. A few examples will suffice. The Japanese Commission ( 1 9 1 ) laid down some useful recommendations for practical prevention. According to these, any evidence of the disease should be reported by local officials to the central government office, and competent inspectors should make a physical examination of all horses weekly during the epizootic season and once a month during the rest of the year in regions where infectious anemia is prevalent. Infected animals must be destroyed or isolated, and suspicious cases removed from pastures. If a positive case occurs among a pastured group during the horsefly season, the whole herd should be considered infected. The movement of horses from infected regions must be prohibited. Isolation stables should be located sufficiently distant from the ones used for healthy animals and infected straw, dung, and all such material should be kept in heaps at least for two months and may not be used as manure before the expiration of that period. Scott ( 3 9 6 ) discussed the control measures in 1919, when examining the economic importance of infectious anemia in Wyoming, and advocated killing all affected cases immediately—provided the diagnosis is correct— "since many of the infected horses die anyway and the remainder as carriers.of the virus become a menace to the rest of the herd". The Japanese suggested setting free recovered cases if they are perfectly free of symptoms, but Scott considers this plan as not entirely safe because, in most cases, the blood of such horses is still virulent. If all affected horses are not killed, he suggested the segregation of sick and well horses by removing the latter to a new, dry pasture at a distance far enough to prevent the passage of biting flies that he clearly has shown in his experiments to be potential transmitters of the virus. The safest isolation is in a barn with openings screened. For reducing the incidence as much as possible, it is important that a careful watch be kept over all horses exposed to the infection (checking the animals' temperature and, eventually, their urine for albumen). Movements and shipment of all horses from an area where an outbreak occurs, must be suspended particularly during the infective season; suspicious cases and chronic carriers should not be moved at any time. Disinfection of stables, though desirable from the sanitary point of view, is considered of little value in preventing the spread of infection. Serum companies must always be on guard to detect and avert the presence of virus carriers among their production horses, on account of the risk of spreading the infection by the use of preventive and therapeutic horse serum, or other products. Fortner ( 1 2 0 ) reported that 3000 horses had to be destroyed because of the use of a vaccine (against Borna-disease) made from brain tissue of a horse carrying the virus of infectious anemia He suggested that strict quarantine measures for the horses be put into effect four months before they are used for serum production, during

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which time they should be observed closely and temperatures should be taken daily; any suspicious cases should be checked by the horse inoculation test. He also analyzed the problem of differential diagnosis as presented by minor symptoms seen in some of the hyperimmunized serum horses ( 1 2 1 ) . Rohrer and his co-workers (374,376) recently studied the method of "cross-infection" testing in horses which means that pairs of horses are injected with each other's blood. According to them, this procedure would, in most cases, reveal eventual carriers among horses intended for use in serum production. Although they encountered some difficulties in interpreting their results, they reported finding 3 per cent virus carriers among a group of 126 clinically healthy horses by the cross-infection method. The main difficulty lies in the interpretation of mild reactions, since it is not easy to decide whether the donor's blood or the condition of the recipient animal is responsible for the uncertain symptoms. The United States Bureau of Animal Industry established a safeguard against dissemination of the disease through the use of biological products by requiring that biological supply houses operating under government license heat all antisera prepared from horses at 58° to 59° C. for one hour(426). Studies of the Bureau of Animal Industry, as described by Stein ( 4 2 6 ) , indicate that the following measures are the most effective means of control: When a definite diagnosis of infectious anemia has been made, one should kill the animal and dispose of the carcass by cremation or deep burial in order to prevent further spread of the infection. If it is not feasible to destroy infected animals, then those known to be diseased and those suspected of being infected should be isolated from healthy animals. The common use on both infected and healthy horses of equipment that may produce skin abrasions or absorb body excretions or secretions, such as bridles, harnesses, saddles, blankets, brushes, and currycombs is to be avoided. Surgical instruments, needles and syringes should be thoroughly sterilized for each animal. Infected mares should not be used for breeding purposes, as they may transmit the disease to their offspring. Animals should not be kept together in small, poorly drained paddocks adjacent to stables and manure dumps. Where premises are badly contaminated, or a number of cases of the disease have developed on certain pastures, it is first advisable to move the animals to new quarters, then plow the ground deeply and fence off the infected area. Horses or mules should not be permitted on such pastures or premises for at least 6 months. There should be maintained good sanitary conditions, fly control, systematic control of intestinal parasites, and a supply of pure, fresh drinking water; under no circumstances should animals be permitted to drink from stagnant pools. The procedure carried out in New Hampshire, according to the 1948 report of the Chief of the Bureau of Animal Industry ( 4 6 2 ) , for controlling

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EQUINE INFECTIOUS A N E M I A

the spread of the infection during the epizootic of 1947 in New Hampshire among a group of thoroughbred horses was as follows: A n 8 6 - d a y quarantine was placed o n the h o r s e s at R o c k i n g h a m Park and surrounding territory. All sick horses w e r e isolated and placed in an isolation unit. T e m p e r a t u r e s of horses at the track stables w e r e taken daily to detect early cases. F l i e s w e r e controlled by the use of effective fly repellents. Separate drinking and f e e d i n g facilities w e r e provided f o r each horse, and the barns and i m m e d i a t e surr o u n d i n g s w e r e kept in g o o d sanitary c o n d i t i o n . All straw, m a n u r e , and o t h e r r e f u s e f r o m the isolation unit w e r e destroyed by burning. B e f o r e the quarantine w a s lifted the horses that had s h o w n clinical s y m p t o m s of the disease w e r e d e s t r o y e d and the s u p p o s e d l y n o r m a l horses were tested by a 4 0 - d a y horse i n o c u l a t i o n test.

For reducing the chances of the spread of infection by insects, either careful screening, or spraying of the stables is recommended with D D T or other insecticides ( 4 3 5 ) . We utilized both of these measures during our experiments. Reports have been published concerning the successful eradication of the disease from regions where it existed before. H o m e ( 1 7 2 ) stated that strict government control measures added much to practical eradication in Norway. According to Martini ( 2 8 1 ) , such measures were successful in Slovenia. Fairly good success was claimed also as a result of enforcing official control measures in Prussia ( 2 0 2 ) . Although several papers have been published about infectious anemia that was enzootic among small African donkeys in South Africa, the disease is not even mentioned in Henning's book published in 1949 on Animal Diseases in South Africa. It would seem that the disease has disappeared since the early experimental studies were performed. The difficulties of eradicating the disease from a certain area where it has been enzootic for a long time are almost insuperable for the simple reason that tracing all of the healthy-looking symptomless virus carriers is practically impossible without an inexpensive method for detecting such horses. As has been discussed in the chapter dealing with the problem of immunization, preventive vaccination has been studied by many investigators with very little success so far. Thus, it is not included among the list of practicable control measures for the purpose of checking the spread of infection.

CONCLUDING REMARKS It seems appropriate, in conclusion, to add a few words to what is said in the preceding chapters about the original targets of our experimental studies and the results achieved. We laid the emphasis in our studies on the experimental aspects. The clinical side of the disease could not be treated equally well, partly for lack of time, and partly because the clinical aspects are better known and described. It is obvious that what is lacking for our better understanding of this malady can be best learned by experimental approach, that is, by the application of methods of investigation developed in recent years for the clarification of similar problems. This explains why, throughout the discussions in this work, preference is given to facts learned by experimentation. By necessity, the scope of the experimental studies was a modest one, for a great variety of problems had to be explored by a small group. Obviously, a larger group of investigators, representing several experimental fields, could attack more successfully such complex problems as we dealt with. It seemed desirable for us to explore simultaneously as many phases of the problem as possible, although the number of the experimental horses that we were able to handle at the same time was limited. This made it necessary to limit the scope of any given experiment, in order to turn to the exploration of the next problem. Studying one single problem for a long time would have prevented us from looking into a series of diverse questions which all seemed to be important if we wanted to make progress in our investigations. Consequently, several of the problems that we tried to explore experimentally were not studied in sufficient detail, as we were not able to carry them to a firm conclusion during the few years while this experimental work was in progress. It is felt, however, that in spite of the rather limited extent to which these experiments could be developed, a definite progress was made and several unsolved problems concerning equine infectious anemia came closer to a possible solution. Our main targets in the experimental studies were attempts at transmission of the infection to small laboratory animals, developing laboratory tests for a quick diagnosis, and eventually finding means of preventive immunization against the disease. The latter two could be best achieved by successful cultivation of the virus outside of the animal's body. As briefly (145)

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outlined in the corresponding chapters of this monograph, in several phases of our work promising results were recorded which—we believe—may serve as a basis for further attack on the problems. Systematic experiments conducted to clarify the nature of the pathologic process disclosed the fact, as outlined in Chapter 9 (page 7 1 ) , that in infectious anemia the primary infection with the virus leads to an allergic state in the infected animal and is not followed by immunity observed in many other infectious diseases. Rather it makes the animal more sensitive to renewed contact with the virus. This explains many strange findings noted by other experimenters as well as by ourselves in studying infectious anemia. It also explains the very limited success noted so far in attempts at preventive immunization. We believe that, even after successful and proven cultivation of the virus, the complicating factor of hypersensitivity present in the infection must be overcome by some device of desensitization before immunization can be achieved. We are fully aware that in our discussions we often raised more questions than we have answered. It is felt, however, that the weighing of theoretical possibilities may be helpful in future investigations. We are confident that the expansion of our findings regarding the allergic mechanism of the infectious process will contribute to the clarification of the "mystery" of infectious anemia. This monograph is, naturally, not intended to present the solution to the problems of infectious anemia. It is rather to provide orientation for those problems. Our conclusions in the various chapters are intended to be helpful in formulating opinion on the material presented. As stated in the Foreword, we had the task of bringing together the pertinent information on the subject of infectious anemia and presenting it in concise form. In doing so we divided the material according to subject matter closest to the respective fields of the authors. Dr. Lombard wrote the chapter on Histopathology; she also prepared Chapters 3, 4, and 14 and contributed to the compilation of Chapter 7. F o r the remaining chapters and for the interpretation of the experimental findings, the senior author has to take the responsibility. The bibliography at the end of this monograph lists publications up to the end of 1951. For the sake of brevity, we mentioned the most needed references in the discussions of the various chapters. These may well serve for first orientation and may form the basis for further search in the literature on the particular subject. To obtain the full story, the rich material listed in the supplementary bibliography should also be consulted. In reporting developments, we tried to respect priority as much as possible, although our primary object was not to analyze the voluminous literature in detail.

CONCLUDING

A CKNO

REMARKS

147

WLEDGMENTS

It is our pleasant duty to thank the many friends and colleagues who by their unselfish cooperation helped us in performing our experimental work and compiling the present report about it. We wish to express our gratitude to the late Dr. Raymond A. Kelser, to whose memory this monograph is dedicated, for his inspiring guidance, understanding, and warmhearted encouragement throughout the many difficult phases of the work. His enthusiasm was always an inspiration to his associates. We are deeply indebted to Dr. Richard E. Shope for his constant interest in our work and for his many useful suggestions both in the experimental work and in the preparation of this monograph. We feel that without his support and understanding this report could not have been prepared. Our heartfelt thanks are due to Dr. Evan L. Stubbs, head of the Department of Pathology, who obligingly offered to us his help and that of his entire staff in making post-mortem investigations of the experimental horses, and in handling and preparing practically all of the histologic material used in our study. We are grateful to Dr. J. D. Beck, Dr. A. G. Danks, Dr. Th. DeMott, and Dr. W. E. LaGrange for their valuable help and assistance in making the facilities of Bolton Farm available; and to Dr. I. Live and Dr. M. J. Deubler of the Department of Microbiology for various services while we carried out our laboratory work. It is impossible to thank, by name, our many friends and colleagues who willingly helped us during our work, with advice and otherwise, because we would be compelled to list most of the members of the Faculty. We offer our best thanks to them. We are indebted to Dr. John W. Mills, of the Veterinary School, and Dr. Steven C. Mohos of Cornell University Medical College for the fine microphotographs used for illustration of the monograph, and to Dr. Mohos for his comments regarding the interpretation of the microscopic findings. Finally, we want to express our best appreciation to our co-workers whose names do not appear on the cover of the present report but who made valuable contributions to our experimental work, while they participated in various phases of the study. Our thanks are due to Dr. Werner Leemann of the University of Zurich, Switzerland, to Dr. Arnold R. Gilman, and Miss Jane F. Shirer. Dr. Gilman's well-planned and ambitious work and its findings constitute an integral part of the study. Miss Shirer has been associated with us almost during the whole tenure of our studies, assisting with the experiments and also typing and checking the list of bibliography; her skillful and conscientious work was most valuable. We are grateful to Mrs. Mary McK. Wildermuth of the University of Pennsylvania Press for checking our manuscript and suggesting many improvements and corrections in English. We are indebted to the Grayson Foundation for sponsoring the project and providing the necessary financial support for the experimental investigation.

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E Q U I N E INFECTIOUS A N E M I A L I S T OF ABBREVIATIONS

(Journals Allatorv.Lapok Amer.J.Vet.Med. Amer.J.Vet.Res. Amer.Vet.Rev. Arch.ges.Virusforsch. Arch.Path. Arch.Tierheilk. Berl.-Munch.Tier.Woch. Berl.Tier.Woch. Bull. Acad. Vet.France BuII.Off.Intl.Epizoot. BuIl.Soc.Centr.Med.Vet. Canad.J.Comp.Med. Compt.Rend.Acad.Sci. Compt.Rend.Soc.Biol. Cornell Vet. Deut.Tier.Woch. Exp.Sta.Rec. Jap.J.Vet.Sci. J.Amer.Med.Ass. J.Amer.Vet.Med.Ass. J.Exp.Med. J.Immunol. J.Lab.Clin.Med. Jber.Vet.Med. Maanedsskr.Dyrlaeg Monat.Tierheilk. Monat.Vet.-Med. Munch.Tier.Woch. Nord.Vet.Med. Norsk VetTidsskr. North Amer.Vet. Proc.Soc.Exp.Biol.Med. Rec.IVTed.Vet. Rev.Gen.Med.Vet. Rev.Med.Vet. Schweiz.Arch.Tierh. Skand.VetTidskr. Tidskr.Vet.-Med. Tierarzt.Rund. Tijdschr.Diergeneesk. Vet.BuIl. Vet.Med. Vet.Journ. Virchow's Arch.Path.Anat. Wien.Tier.Monat. Z.Infekt.Haustiere Z.Vet.kunde

most frequently

quoted in the

Bibliography)

Âllatorvosi Lapok American Journal of Veterinary Medicine (later Vet.Med.) American Journal of Veterinary Research American Veterinary Review (later J.Am.Vet.Med.Ass.) Archiv für die gesamte Virusforschung Archives of Pathology (and Laboratory Medicine) Archiv für wissenschaftliche und praktische Tierheilkunde Berliner und Münchener tierärztliche Wochenschrift Berliner tierärztliche Wochenschrift Bulletin de l'Académie vétérinaire de France Bulletin. Office international des épizooties Bulletin et mémoires. Société centrale de médecine véterin. Canadian Journal of Comparative Medicine and Veterinary Science Comptes rendus hebdomadaires des séances de l'Académie des Sciences (Paris) Comptes rendus hebdomadaires des séances et mémoires de ta Société de Biologie (Paris) Cornell Veterinarian Deutsche tierärztliche Wochenschrift Experiment Station Record Japanese Journal of Veterinary Science Journal of the American Medical Association Journal of the American Veterinary Medical Association Journal of Experimental Medicine Journal of Immunology Journal of Laboratory and Clinical Medicine Jahresbericht über die Leistungen auf dem Gebiete der Veterinär-M edizin Maanedsskrift for Dyrlaeger Monatshefte für praktische Tierheilkunde Monatshefte für Veterinärmedizin Münchener tierärztliche Wochenschrift Nordisk Veterinaermedicin Norsk Veterinaertidsskrift North American Veterinarian Proceedings, Society for Experimental Biology & Medicine Recueil de médecine vétérinaire de l'École d'Alfort Revue générale de médecine vétérinaire Revue de médecine vétérinaire Schweizer Archiv für Tierheilkunde Skand in avis k V eterinärtidskrift Tidskrift for Veterinär-Medicin Tierärztliche Rundschau Tijdtchrifl voor Diergeneeskunde Veterinary Bulletin Veterinary Medicine Veterinary Journal Virchonu's Archiv f . pathologische Anatomie u. Physiologie Wiener tierärztliche Monatsschrift Zeitschrift für Infektionskrankheiten der Haustiere Zeitschrift für Veterinärkunde

BIBLIOGRAPHY A) REFERENCES (Throughout

the text, numbers in parentheses

refer to publications

listed

below)

1. Abbondanza, S.: E una leptospirosi l'anemia infettiva dei cavalli della zona risiera Lombardo-Piemontese? Clinica Vet. 1951, 74. 10 2. Abderhalden, E., und Frei, W . : Ueber das Verhalten des Blutes (Plasma resp. Serum und rote Blutkörperchen) von an perniziöser Anämie erkrankten Pferden gegen Saponin. Arch. Tierheilk. 1910, 36. 423 3. Akerström, R.: Sublingual hemorrhage in infectious anemia in horses. Nord. Vet.Med. 1951, 3.- 659 4. Alegren, A.: Anemie infectieuse du cheval en Suède. Bull. Off. Intl. Epizoot. 1951, 36. 312 5. Altara, I . : L'anémie infectieuse des équidés. (Epizootologie de la maladie.) Bull. Off. Intl. Epizoot. 1951, 36. 232 6. Andrewes, C. H . : Interference by one virus with the growth of another in tissue culture. Brit. J. Exp. Path. 1942, 23. 214 7. Andrievskii, I . : Immunity in equine infectious anemia. {In Russian) Sovyet Vet. 1939, 16. (No. 8) 25 (abstr.: V.B. 10. 671) 8. A n g i n i a r d : Observations de contagion par infection de l'anhémie idiopathique du cheval. Rapport de la commission. Ree. Méd. Vét. 1859, 36. 639 Discussion: 1861, 38. 325, 421 9. Aschoff, L.: Zur Myocarditisfrage. Verhandl. Dent. Path. Ges. 1904, 8. 46 10. Baker, J. A.: Rinderpest. VIII. Rinderpest infection in rabbits. Amer. J. Vet. Res. 1946, 7. 179 11. Balozet, L.: Etude clinique et expérimentale de l'anémie infectieuse des équidés au Maroc. Bull. Soc. Path. Exot. 1923, 16. 525 12. Balozet, L. : Au sujet de l'immunité dans l'anémie infectieuse des équidés. Compt. Rend. Acad. Sci. 1934, 198. 992 NOTE.—BOLDFACE numbers indicate volume of journal. Abbreviations used when referring to an abstract of original papers: B.T.W. B.M.T.W. D.T.W. J.A.V.M.A. Jber. M.T.W. V.B. Diss.

Berliner Tierärztliche Wochenschrift Berliner und Miinchener Tierärztliche Wochenschrift Deutsche Tierärztliche Wochenschrift Journal of the American Veterinary Medical Association Jahresbericht über Veterinär-Medizin Miinchener Tierärztliche Wochenschrift Veterinary Bulletin (Brit.) designates doctoral dissertation, followed by. the name of city where it was submitted as a thesis

(149)

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683. G o s s l e r , G . : D e r W e r t des K a n i n c h e n i m p f v e r s u c h e s f ü r die D i a g n o s e d e r i n f e k t i ö s e n A n ä m i e des P f e r d e s . Diss. H a n n o v e r , 1934 ( a b s t r . : J b e r . 56. 106) 684. G o u b i n e : Les p o r t e u r s et les e x c r é t e u r s du v i r u s de l ' a n é m i e i n f e c t i e u s e d u cheval. (In Russian) T r u d . Vsesoyuz. I n s t . E k s p . Vet. 1936, 12. 30 ( a b s t r . : Ree. M é d . Vét. 113. 618) 685. G o u r v i t c h , B. M., Oleinik, K . K . , a n d A v r o m e n k o , G . D . : T h e c o m p l e m e n t fixation test in equine i n f e c t i o u s a n e m i a . (In Russian) Sovyet Vet. 1936, 13. ( N o . 6) 24 686. G ö v e r t , H . : W e i t e r e U n t e r s u c h u n g e n ü b e r die D i a g n o s e d e r a n s t e c k e n d e n B l u t a r m u t des P f e r d e s m i t H i l f e d e r v e r e i n f a c h t e n K a n i n c h e n v e r s u c h e s . D i s s . H a n n o v e r , 1938 ( a b s t r . : J b e r . 63. 384) 687. G r a t z l , E., u n d T r a u b , E . : Z u m V o r k o m m e n v o n h ä m o l y t i s c h e n S t r e p t o k o k k e n bei d e n k a t a r r h a l i s c h e n t z ü n d l i c h e n E r k r a n k u n g e n d e r L u f t w e g e des P f e r d e s . A r c h . T i e r h e i l k . 1942, 77. 347 688. G r a y s o n F o u n d a t i o n : C u r r e n t r e s e a r c h p r o j e c t s . E q u i n e i n f e c t i o u s a n e m i a . Bulletin of G r a y s o n F o u n d a t i o n ( p . 14). N e w Y o r k , 1948 689. G r a y s o n F o u n d a t i o n : S t a t u s of r e s e a r c h p r o j e c t s . E q u i n e i n f e c t i o u s a n e m i a . Bulletin of G r a y s o n F o u n d a t i o n ( p . 1 2 ) . N e w Y o r k , 1950 690. G r e e n b e r g , D . M., a n d M i r o l u b o v a , T . N. : M o d i f i c a t i o n s in the c o l o r i m e t r i c d e t e r m i n a t i o n of the p l a s m a p r o t e i n s by the Folin p h e n o l r e a g e n t . J . L a b . Clin. M e d . 1936, 21. 431 691. G r e s s n e r , J . : H i s t o p a t h o l o g i c a l c h a n g e s of the spleen in i n f e c t i o u s a n e m i a . (In Czech) T h e s i s , B r n o , 1935 ( a b s t r . : J b e r . 60. 309) 692. Griffin, C. A., a n d Brose, C. P . : R e p o r t of an o u t b r e a k of e q u i n e i n f e c t i o u s a n e m i a w i t h o b s e r v a t i o n s on blood c h a n g e s . J . A m e r . Vet. M e d . A s s . 1936, 89. 664 693. G r o h t , W . : U e b e r B l u t b e f u n d e bei mit i n f e k t i ö s e r A n ä m i e b e h a f t e t e n P f e r d e n , anschliessend p r o p h y l a k t i s c h e V e r s u c h e m i t B 205 u n d B 844. D i s s . H a n n o v e r , 1924 ( a b s t r . : J b e r . 44. 92) 694. G r u n e r t , C. H . : N e w s u g g e s t i o n s on s w a m p f e v e r t h e r a p y . Vet. M e d . 1925, 20. 15 695. Guillot, G., et C a u d r o n , M . : L a r é a c t i o n de F u l t o n et la r é a c t i o n au c a r m i n ( C h o r i n e ) . R e v . Vét. M i l i t . 1939, 23. 149 696. G u n n e m a n n , F . : I n f e c t i o u s a n e m i a in t h e P a j a l a d i s t r i c t . (In Swedish) Svensk V e t T i d s k r . 1931, 36. 310 697. G u n n e m a n n , F . : Some e x p e r i e n c e w i t h p e r n i c i o u s a n e m i a in h o r s e . (In Swedish) S k a n d . V e t T i d s k r . 1939, 29. 133 698. G u t s c h e , W . : D i e a n s t e c k e n d e B l u t a r m u t . Z. V e t . k u n d e 1919, 31. 221 699. H a a g , A . : B e i t r a g z u r D i a g n o s e d e r a n s t e c k e n d e n B l u t a r m u t des P f e r d e s . Diss. M ü n c h e n , 1923 ( a b s t r . : M . T . W . 75. 1087) 700. H a b e r m a l z , F . : V e r s u c h e ü b e r die A b t ö t u n g des V i r u s d e r i n f e k t i ö s e n A n ä m i e d e r P f e r d e im S e r u m d u r c h F o r m a l i n . Diss. H a n n o v e r , 1924 ( a b s t r . : D . T . W . 32. 759) 701. H a b e r s a n g : B e i t r a g z u r Biologie des E r r e g e r s d e r i n f e k t i ö s e n A n ä m i e d e r P f e r d e . M o n a t . T i e r h e i l k . 1921, 32. 1 702. H a b e r s a n g : Z u r a n s t e c k e n d e n B l u t a r m u t d e r P f e r d e . B e r l . T i e r . W o c h . 1924, 40. 281 703. H a b e r s a n g , F . : I s t die i n f e k t i ö s e A n ä m i e d e r P f e r d e ein u n l ö s b a r e s P r o b l e m f ü r die S e u c h e n b e k ä m p f u n g ? T i e r ä r z t . M i t t . 1937, 18. 755 704. H a b e r s a n g : U e b e r die P r o v o k a t i o n d e r i n f e k t i ö s e n A n ä m i e d e r P f e r d e d u r c h S C V G a s b e h a n d l u n g . M o n a t V e t . - M e d . 1950, 5. 289 705. I l a d w e n , S.: N o t e s on s w a m p f e v e r . J . A m e r . Vet. M e d . A s s . 1920, 56. 439 706. H a d w e n , S., a n d B r u c e , E. A . : A n a p h y l a x i s in c a t t l e a n d s h e e p p r o d u c e d by the l a r v a e of tlypoderma bovis, H. Uneatum a n d Oestrus o