COMPARATIVE STUDIES OF ilNFECTIOUS ANEMTAS IN RATS Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY SANTI THOONGSUWAN 1976 i , L I B R A R Y Michigan State University This is to certify that the thesis entitled OOHPLRATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS presented by Santi Thoongsuwan has been accepted towards fulfillment of the requirements for Phone degree in membiology fihflMj/XJ Wok Major professor ~ e.e. - -— « Herbert W. Cox Dateb (/15; Q 9: “‘12 C Professor , Department of Microbiology and Public Health , Michigan State University. 0-7639 ABSTRACT COMPARATIVE STUDIES or INFECTIOUS ANEMIAS IN RATS By Santi Thoongsuwan A syndrome of anemia with splenomegaly and nephritis accom- panied by autoimmune factors was associated with infections of unre- lated agents. It was called the "anemia-big spleen syndrome" by those relating it to immunoconglutinin (1K), and the "infectious anemia syndrome" by others relating it to cold-active hemagglutinin (CAH). This dissertation reports on a study of the immunopathologic factors associated with infectious anemia in rats resulting from infections of filterable agent of rat infectious anemia (RIA), Trypanosoma lewisi, Babesia rodhaini and Plasmodium chabaudi. The agent of RIA was discovered as a contaminant of a I, lewisi infection. It was less than 0.20 micron in size and in mature rats it caused fatal hemolytic anemia with splenomegaly and glomeruloneph- ritis associated with CAH. Weanling rats recovered after less severe disease. While the agent was not cultured or visualized, its Size and the absence of other agents led to the suggestion that it was a virus. A new strain of I, lewisi was proven to be free of RIA agent. A less severe anemia with splenomegaly and glomerulonephritis accompanied by CAH was found to be associated with infection of this parasite. .E- Santi Thoongsuwan rodhaini and E, chabaudi had been previously associated with infectious anemia. Serum from rats made anemic by each of the infections contained elevated titers of both CAH and IK. It therefore appeared that the anemia-big spleen and infectious anemia syndromes were essentially the same disease. Serum of rats recovered from infectious anemia contained IK but little or no CAH. When challenged with a heterologous agent they exhibited a nonspecific acquired resistance manifested by early anemia, reduced parasitemia, increased titers of IK and CAH and enhanced sur- vival. While the resistance was associated with IK, it was also associated with CAH and antibody to soluble serum antigen (ABSA). Unin- fected rats with autostimulated IK also exhibited the resistance after challenge. Autostimulated rat IK, detected by agglutination of complement- fixed sensitized sheep erythrocytes, was IgM which reacted specifically with fixed C3. The IK levels reached peak values during the acute phase of infection. Chromatography of CAH-free hyperimmune rat globulin separated IK with the 19 S and ABSA with the 7 8 fractions and indicated that CAH, IX and ABSA were different antibodies. The use of these fractions and the whole immune globulin to passively immunize rats infected with RIA agent and mice infected with E, chabaudi indicated that IK alone did not furnish protection, but animals given ABSA exhibited the early anemia, reduced parasitemia and prolonged survival. Whole globulin, containing both IK and ABSA, enhanced this effect of nonspecific acquired resistance. Santi Thoongsuwan Fluorescein conjugated ABSA and IX both reacted with erythro- cytes of blood films and spleen impression slides from rats made anemic by each infection. ABSA was considered to react with soluble serum antigen (SA) which bound as a complex with its antibody to the cells. The IK reaction was believed to be with complement that had been fixed by the complex. It appeared that complexes of SA and ABSA had roles in both anemia and nonspecific acquired resistance and the action might be enhanced by IK. In binding to blood cells or parasites the complexes could mimic opsonin to cause splenic sequestration or hemolytic crisis if optimal complement fixation by the complex was attained. The con- glutinating activity of IK could enhance phagocytosis and sequestration. In animals that had IK and ABSA before SA was elaborated from infection, the complexes would form earlier and be removed from the circulation before they attained dangerous titers. Thus while parasites as well as blood cells were reduced by this nonspecific acquired resistance, it was suggested that the major protective function was primarily the early removal of immune complexes and keeping their titers below a critical threshold. COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS BY Santi Thoongsuwan A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1976 Dedicated to my wife, Mrs. Yanee Thoongsuwan and my family ii ACKNOWLEDGMENTS I wish first to express my appreciation for the doctoral fellowship awarded to me from the Chulalongkorn University, Bangkok, Thailand, that made it possible for me to continue my program of advanced studies in the United States of America and in the Department of Microbiology and Public Health at Michigan State University. I wish to express my sincere appreciation to my academic advisor, Dr. Herbert W. Cox, for his guidance and encouragement throughout these studies and during the preparation of this disser- tation. Advice and helpful criticism of the dissertation received from my guidance committee, Dr. Gordon R. Carter, Dr. Richard A. Patrick, Dr. Jeffrey F. Williams, and Dr. Harold D. Newson, are gratefully acknowledged. I wish also to acknowledge the contribution of Dr. Norman B. McCullough who served on my guidance committee in the absence of Dr. Carter, and of Dr. Robert R. Brubaker and Dr. Ralph N. Costilow who served as departmental representatives for my exami- nations. In addition, I wish to thank Dr. Patrick for his advice, technical assistance and for the use of his laboratory facilities which allowed me to accomplish a part of my research. Technical assistance received from Dr. C. Wayne Smith, Dr. David Liu, iii Mr. A. Wayne Roberts and Mrs. Barbara H. Giese was gratefully appre- ciated. I deeply appreciate the encouragement and moral support of Miss Pisawat Dutiyabodhi, Chairperson of the Department of Microbiology at the Faculty of Pharmacy, Chulalongkorn University. Without her, I may have never had the opportunity of undertaking these studies. I also wish to thank Mrs. Lacy C. Cox for her personal kindness and moral support. The companionship of my fellow students, William J. Rickman, Daniel J. Murfin, and Janet M. Szabo, will be remembered with affection. I wish to acknowledge the support from the Michigan Agricul- tural Experiment Station and the General Research Funds from the College of Veterinary Medicine of Michigan State University which made it possible to carry on this research. iv TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . x INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3 Definition of Infectious Anemia . . . . . . . . . . . . . . . 3 Premunition and Infectious Anemia . . . . . . . . . . . . 4 Autoimmune Phenomena in Infectious Diseases . . . . . . . . . 9 Antigen and Antibody Complexes in Diseases . . . . . . . . . 18 The Agents of Infectious Anemia . . . . . . 20 Agents of Infectious Anemia Used for Comparative Study . . . 22 History and Taxonomy of the Agents Studied . . . . . . . . . 25 Filterable Rat Infectious Anemia (RIA) Agent . . . . . . 25 Trypanosoma lewisi . . . . . . . . . . . . . . . . . 25 Plasmodium chabaudi . . . . . . . . . . . . . . . . . . . 29 Babesia rodhaini . . . . . . . . . . . . . . . . . . . . 31 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 ARTICLE 1 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. I. HEMOLYTIC ANEMIA AND GLOMERULONEPHRITIS ASSOCI- ATED WITH HEMAGGLUTININ IN RATS INFECTED WITH A FILTERABLE AGENT . . . . . . . . . . . . . . . . . . . 44 ARTICLE 2 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. II. AUTOIMMUNE-LIKE ANEMIA ASSOCIATED WITH TRYPANOSOMA LEWISI INFECTION . . . . . . . . . . . . . 79 ARTICLE 3 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. III. IMMUNOCONGLUTININ ASSOCIATED WITH ANEMIA AND NONSPECIFIC ACQUIRED RESISTANCE 0F RECOVERED RATS . . 95 ARTICLE 4 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. IV. STUDIES OF THE NATURE OF IMMUNOCONGLUTININ ASSOCIATED WITH INFECTIOUS ANEMIA OF RATS AND ITS ROLE IN NONSPECIFIC ACQUIRED RESISTANCE . . . . . 121 Table LIST OF TABLES Page ARTICLE I COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. I. HEMOLYTIC ANEMIA AND GLOMERULONEPHRITIS ASSOCIATED WITH HEMAGGLUTININ IN RATS INFECTED WITH A FILTERABLE AGENT Mean number of red blood cells persmm3 (RBCSx 106), mean number of trypanosomes per mm (T x 10 ) and standard error of the mean (S.E.) in blood of mature rats infected with the Microbiology and Public Health (MPH) and American Type Culture (ATC) strains of Trypanosoma lewisi . . . . . . . . . . . 65 Mean and standard error of the mean (S. E. ) of red blood cells per mm3 (RBC x 106 ) and the mean and S. E. of the number of trypanosomes per mm3 (T x 10 ) in normal mature control rats, and mature rats recovered from infection with the American Type Culture (ATC) strain of Trypanosoma lewisi, after infection with the Microbiology and Public Health (MPH) strain of I, lewisi . . . . . . . . . . . . 66 Mean number of red blood cells per mm3 (RBC x 106) and standard error of the mean (S.E.) of 8 mature rats after inoculation with the supernatant of freeze-thaw treated whole blood of rats infected with TFA that had been passed through a 0.20 micron (200 nm) Millipore membrane, and 8 mature rats inoculated with the supernatant that passed through a 0.45 micron (450 nm) membrane . . . . . . . . . 67 Mean and standard error of the mean (S. E. ) of red blood cells per mm3 3(RBC x 186 ), mean number of trypanosomes per mm3 (T x 10 ) and the mean titres of cold- active haemagglutinin (CAH) in blood of young rats injected with normal rat blood cell suspension (Group I), and rats infected with TFA (Group II) . . . . . . . . . . . . . . . . . . . . . 68 vi Table Page Mean red blood cell counts (RBC x 106), 1 standard error of the mean (S.E.) and mortality among 8 young rats and 8 mature rats after infection with TFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Average spleen volume in ml of normal rats, rats infected with the Microbiology and Public Health (MPH) strain of Trypanosoma lewisi, and rats infected with TFA found as a companion of the MPH strain of I, lewisi . . . . . . . . . . . . . . . . . . 70 ARTICLE 2 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. II. AUTOIMMUNE-LIKE ANEMIA ASSOCIATED WITH TRYPANOSOMA LEWISI INFECTION The means of spleen volumes, scores of kidney damage (SKD) and of the number of nuclei in the glomerular tuft (NGT) of 8 normal rats and 8 rats infected for 10 days with Trypanosoma lewisi . . . . . . . . . . . . . . . . . . . . . . . . . . 90 ARTICLE 3 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. III. IMMUNOCONGLUTININ ASSOCI- ATED WITH ANEMIA AND NONSPECIFIC ACQUIRED RESISTANCE OF RECOVERED RATS Immunoconglutinin (IK) and cold-active haemag- glutinin (CAH) titres of pooled serum from 5 rats with acute and 5 recovered rats infected with rat infectious anaemia (RIA) agent, Babesia rodhaini, Plasmodium chabaudi and Trypanosoma lewisi . . . . . . . . . . . . . . . . . . . . 112 Mean 1 standard error of erythrocyte counts per mm3 (RBC x 106 i S.E.), mean titres of immuno- conglutinin (1K) and cold-active haemagglutinin (CAH) and mortality among control rats, recovered from infections of Trypanosoma lewisi and Babesia rodhaini after challenge with rat infectious anaemia agent . . . . . . . . . . . . . . . . . 113 Megn i standagd error of erythrocyte counts per mm (RBC x 10 i S.E.), the percentage of para- sitized erythrocytes (% PE i S.E.), mean titres of immunoconglutinin (IR) and cold-active haemag- glutinin (CAH) and mortality among 25 control rats and 25 recovered from rat infectious anaemia (RIA) after challenge with Babesia rodhaini . . . . . . . . 115 vii Table Page Mean i standard error of erythrocyte counts per mm3 (RBC x 106 i S.E.), % parasitized erythro- cytes (% PE i S.E.), mean titres of immunocon glutinin (IK) and cold-active haemagglutinin (CAH) and mortality among 25 control rats and 25 recovered from rat infectious anaemia (RIA) after challenge with Plasmodium chabaudi . . . . . . . . . . . . 117 Mean number of red blood cells persmm3 (RBCSX 106), mean number of trypanosomes per mm (T x 10 ) and standard error of the mean (S.E.) in blood of 6 control rats (Control) and 6 rats recovered from rat infectious anaemia (RIA-Recovered) after challenge with Trypanosoma lewisi . . . . . . . . . . . . . 119 Mean i standard error of the mean (S.E.) of red blood cells per mm3 (RBC x 106), percentage of parasitized erythrocytes (% PE), mean titres of immunoconglutinin (IK) and cold-active haemag- glutinin (CAH) of normal rats and rats immunized with autologous normal rat serum absorbed on kaolin (SAX) after Babesia rodhaini infection . . . . . . . 120 ARTICLE 4 _ COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS 2A. IN RATS. IV. STUDIES OF THE NATURE OF IMMUNOCONGLUTININ ASSOCIATED WITH INFEC- TIOUS ANEMIA OF RATS AND ITS ROLE IN NONSPECIFIC ACQUIRED RESISTANCE Mean erytgrocyte counts per mm3 1 standard error (RBC x 10 i S.E.) of uninfected rats injected with whole globulin from rats hyperimmunized by Babesia rodhaini infections, and of rats infected with rat infectious anaemia agent which were then injected with normal rat serum (NRS), with 198 (IK) fraction of hyperimmune globulin, with 78 (ABSA) fraction of hyperimmune globulin and with whole hyperimmune globulin . . . . . . . . . . . . . . . . 139 Mean erythrocyte counts per mm3 : standard error (RBC x 106 t S.E.) of uninfected mice injected with whole globulin from rats hyperimmunized by Babesia rodhaini infections, and of mice infected with Plasmodium chabaudi which were then injected with normal rat serum (NRS), with 198 (IK) frac- tion of hyperimmune globulin, with 78 (ABSA) fraction of hyperimmune globulin and with whole hyperimmune globulin . . . . . . . . . . . . . . . . . . . 141 viii Table Page 28. Mean percentage of parasitized erythrocytes 1 standard error (% PE i S.E.) of mice in the same experiment as Table 2A . . . . . . . . . . . . . . . . . . 143 ix LIST OF FIGURES Figure Page ARTICLE 1 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. I. HEMOLYTIC ANEMIA AND GLOMERULONEPHRITIS ASSOCIATED WITH HEMAGGLUTININ IN RATS INFECTED WITH A FILTERABLE AGENT 1. Photomicrographs of blood films and spleen impression slides from normal rats and rats with TFA infection after incubation with FITC conjugated globulin from rats hyperimmunized by TFA infections . . . . . . . . . . . 72 2. Photomicrographs of Giemsa stained spleen impression slides (lOOOX) . . . . . . . . . . . . . . . . . . . . . . 74 3. Photomicrographs of Giemsa stained bone marrow films (lOOOX) . . . . . . . . . . . . . . . . . . . . . . 4. Photomicrographs of rat kidney sections cut at 4 u and H and E stained (700X) . . . . . . . . . . . . . . . . 78 ARTICLE 2 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. II. AUTOIMMUNE-LIKE ANEMIA ASSOCIATED WITH TRYPANOSOMA LEWISI INFECTION 1. Mean and standard error of the mean for erythrocyte counts (RBC x 106), trypanosome counts (trypanosomes x 105) and titers of cold-active haemagglutinin (HA titer) in rats after infection with 1 x 10 Trypanosoma lewisi parasites and in control rats injected with l x 10 normal rat erythrocytes . . . . . . . 92 2. Photomicrographs of sections of kidneys from rats infected for 10 days with Trypanosoma lewisi and from a normal rat . . . . . . . . . . . . . . . . . . . . . 94 Figure Page ARTICLE 4 COMPARATIVE STUDIES OF INFECTIOUS ANEMIAS IN RATS. IV. STUDIES OF THE NATURE OF IMMUNOCONGLUTININ ASSOCIATED WITH INFECTIOUS ANEMIA OF RATS AND ITS ROLE IN NONSPECIFIC ACQUIRED RESISTANCE l. The optical density (0.D.) at 280 nm and the titres of immunoconglutinin (IK) in fraction samples after column chromatography with Sephadex 6-200 of pooled, cold agglutinin-free, globulin from rats with acute Babesia rodhaini infection . . . . . . . . . . . . . . . . 146 2. The optical density (0.D.) at 280 nm, the titres of immunoconglutinin (IK) and the results of tests for antibody to serum antigen (ABSA) in fraction samples after column chromatography with Sephadex 6-200 of pooled globulin from rats hyperimmunized by Babesia rodhaini infections . . . . . . . . . . . . . . 148 3. Photograph of immunodiffusion in gel test of 4S, 19S, and 7S fraction pools obtained by Sephadex G-200 column chromatography of globulin from rats hyper- immunized by Babesia rodhaini infections, whole globulin of the hyperimmunized rats (HIBr), whole globulin from rats hyperimmunized by Plasmodium chabaudi infections (HIPc) and normal rat globulin (N) with serum from rats with acute B, rodhaini infection (Br) . . . . . . . . . . . . . . . . . . . . . . 150 4. Reactions of immunoconglutinin (IK), from 195 fraction pools globulin from rats with acute Babesia rodhaini infection which had been absorbed free of cold-agglutinin, with sheep erythrocytes (E), sensitized sheep RBC (EA), sensitized sheep RBC fixed with C14 (EAC14) and C143 (EAC143) fragments of human complement . . . . . . 152 5. Photomicrographs of immunofluorescent reactions of fluorescein isothiocyanate conjugate of CAH- free IK globulin from rats immunized with autologous serum-absorbed kaolin . . . . . . . . . . . . . 154 6. Photomicrographs of immunofluorescent reactions of fluorescein isothiocyanate conjugated 7S (ABSA) fraction of globulin from rats hyper- immunized by Babesia rodhaini infections . . . . . . . . . 156 xi INTRODUCTION In certain infectious diseases there exists a syndrome of anemia, splenomegaly and glomerulonephritis which is accompanied by the presence of auto-immune—like blood factors. Since the syndrome is more consistently associated with autoimmune factors such as cold- active hemagglutinin (CAH) than to the nature of the infectious agents, it was suggested that the disease was of an autoimmune nature and that it should be referred to as infectious anemia to differ- entiate it from the more classic forms of infectious diseases (Cox and Iturri, 1976). Thus infectious anemia may be associated with the infections of viral, rickettsial, or hemosporidian agents, but have as a common factor the association of CAH with the disease syndrome (Cox and Iturri, 1976; Cox et al., 1966; Oki and Miura, 1970; Schroeder et al., 1966). Infectious anemias differ from the more classic infectious diseases in another respect. Their agents appear to be ineffective as immunizing agents and self curing immune responses to their infections are not conspicuous. It was well recognized by early workers that the acquired resistance of diseases such as malaria differed from the classic form of rejection of the infectious agent associated with sudden cuticular inflammation (allergic inflammation or hypersensitivity) that had been so clearly described by Jenner (1798). They therefore employed the term "premunition" to differentiate the acquired resis- tance of malaria from the classic form described by Jenner. Thus premunition is referred to as the immunity of chronic infection and has been found to be dependent upon the presence of a functional spleen (Sergent, 1963). More recently it was recognized that premunition is in part a phenomenon of nonspecific acquired resistance (F. E. G. Cox, 1972; Cox and Milar, 1968; Ludford et al., 1969). Nonspecific resistance to infectious anemia was generated by immunization with soluble serum antigen (SA) from animals with acute babesial and malarial anemia (Corwin and Cox, 1969; Cox, 1966; Sibinovic et al., 1967). It has therefore been suggested that premunition might be a protective phenomenon active against infectious anemia rather than a resistance against a specific infectious agent (Cox and Milar, 1968). Since CAH was found to be an autoantibody common to infectious anemia associated with a diversity of infectious agents, it is hypoth- esized that SA and other autoantigens might also be common to infec- tious anemia and that premunition might in part be a result of immunization with autoantigens. This hypothesis has been tested in studies of blood factors associated with infectious anemia mediated in rats by a filterable rat infectious anemia agent, babesial, trypanosomal and plasmodial infections. The results of these com— parative studies are presented in this dissertation. REVIEW OF LITERATURE Definition of Infectious Anemia The term "infectious anemia" does not appear in Dorlands, The American Illustrated Medical Dictionary, 20th Edition (1944). It was probably first used by Weinman (1944) in reference to anemia due to infections with Bartonella and related red cell parasites. Since it appeared that Weinman did recognize anemia as a syndrome associated with infections of various agents in man and animals, e.g., Bartonella bacilliformis, Anaplasma marginale, Haemobartonella spp. and Eperythrozoon spp., it is assumed that he had used the term in the sense presently intended. Specifically, infectious anemia refers to a syndrome consisting of anemia with splenomegaly and vascular-renal disease accompanied by autoimmune factors which may be associated with the infections of a variety of unrelated agents (Cox and Iturri, 1976). Thus the syndrome may be associated with the infections of viral, rickettsial or protozoan agents and regardless of the nature of the agent, the essential disease signs will be the same (Cox and Iturri, 1976; Cox et al., 1966; Ludford et al., 1969; McCluskey and Vassalli, 1971; Oki and Miura, 1970; Schroeder et al., 1966; Schroeder and Ristic, 1965). In addition to being the causal agents for the disease syn- drome, the agents of infectious anemia tend to share another common trait in that they appear to be incapable of immunizing their host during infection. Self curing immune responses are therefore not a prominent feature. Acquired resistance associated with these diseases differs from the more classic type demonstrated by Jenner (1798) and has been termed "premunition" to differentiate if from classic acquired immunity. Thus premunition, meaning literally forewarned, has been called infection immunity, or the immunity of chronic infection. Premunition and Infectious Anemia The major in depth treatise on the subject is that of Sergent (1963) which deals primarily with this protective phenomenon as it pertained in experimental and human malaria. It was pointed out that premunition did not produce a blood sterilizing immunity, but in many cases may be effective to the extent that parasitemia may be kept at subpatent levels for indefinite periods. Radical chemotherapeutic cures were soon followed by a complete loss of protection and the maintenance of protection was dependent upon the presence of the spleen. More recent information has indicated that the protection may be of a nonspecific nature and may apply to infectious diseases other than malaria. Rodents that had recovered from babesial infections were resistant to malaria and vice versa (F. E. G. Cox, 1972; Cox and Milar, 1968). Ducklings that had recovered from viral duck infectious anemia were resistant to malarial infections that were lethal for normal ducks (Ludford et al., 1969, 1972). The presence of Eperythrozoon coccoides infections in mice conferred an enhanced resistance to rodent malarias (Ott et al., 1967; Peters, 1965). New Zealand Black (NZB) mice were much more resistant to rodent malaria than were the New Zealand White (NZW) mice, and after recovery from malaria the NZB mice did not show signs of autoimmune anemia and nephritis for more than a year (Greenwood et al., 1970; Greenwood and Voller, 1970a, b; Greenwood and Greenwood, 1971). Since it is now known that NZB mice carry con- genital infections of Gross Lymphoma-Leukemia virus and maintain a persistent viremia (Mellors et al., 1969; Mellors and Huang, 1966), it is possible that while NZB mouse disease may be mediated by factors associated with the viremia, the nonspecific protection associated with these mice might be relatable to the nonspecific phenomenon of premunition. Nonspecific protection was demonstrated against malaria and babesiosis in animals after they had been immunized with soluble serum antigens found in the blood of animals with acute malaria or babesiosis (Corwin and Cox, 1969; Cox, 1966; Sibinovic et al., 1967, 1969). Thus it was indicated that the nonspecific resistance of premunition might be a phenomenon of immunization. The serum antigens associated with malaria and babesiosis were found not to be parasite antigen and the antigen associated with each of the infections were found to be similar if not identical in their serologic reactions. Evidence that these were not parasite antigens led to the suggestion that they might be modified host products or autoantigens (Soni and Cox, 1974, 1975a, b, c). A concept that autoimmune factors relatable to anemia might have a protective function was advanced by Cox (1964) in a discussion of autoimmunity in malaria. It was pointed out that any immune mechanism, autoimmune or otherwise, that had a detrimental effect on erythrocytes might also be harmful to erythrocytic parasites. This overly simplistic concept was tested with the results that it was shown that antibody to normal rat or mouse erythrocytes had a marked deleterious effect on the infectivity of Plasmodium berghei parasites (Cox, 1969). Similar effects were demonstrated in rodent babesiosis and bovine anaplasmosis (McHardy, 1972, 1974). As indicated by Sergent (1963), a functional spleen may be critical to the protection of premunition. Since this organ is richly endowed with cells that participate in the immune response and in immune phagocytosis, and is positioned to be exposed to antigens circulating in the blood, Taliaferro (1956) postulated that the primary penalty of splenectomy in malaria was the loss of the antibody response and the phagocytic activity that the organ contributed to protective immunity. Credibility was given this thought by the demonstration that intact animals responded somewhat better than splenectomized in generating antibody to intravenously administered toxoid (Taliaferro, 1956). However, it is probable that spleen function in premunition is more complex than was indicated by Taliaferro (1956). In discussing hemolytic diseases, Dacie (1962) pointed out that antibody to erythrocytes in the form of cold-active hemagglutinin was frequently found associated with these diseases. He also pointed out that the spleen was one of the primary blood filters of the body and that one of its chief functions was the removal of effete erythrocytes from the blood. He suggested that this function might be enhanced by the action of autohemagglutinins. He speculated further suggesting that congenitally defective erythrocytes or those made defective by infectious agents might have functional antigenic sites that would stimulate production of autohemagglutinin and in turn enhance the removal of these cells from the blood. Thus the specu- 1ations of Dacie (1962) about a protective function for autoantibody against erythrocytes differed from those of Cox (1964) only in that Dacie did not recognize the potential importance of the spleen as a protective process in red cell infections. These thoughts led to experiments in which it was found that cold-active hemagglutinin (CAH) was associated with anemia, spleno- megaly and the phagocytosis of infected as well as uninfected erythro- cytes by phagocytes of the spleen or bone marrow in animals with malaria, babesiosis and anaplasmosis. Since the anemia crisis associ- ated with CAH was accompanied by marked reductions in parasitemia and recovery from malaria and babesiosis, it was suggested that this autoimmunologic process might be related to recovery (Cox et al., 1966; Schroeder et al., 1966). The importance of the spleen in recovery was indicated in that splenectomized bovines had more severe anaplasmosis than intact and often died showing signs of red water disease (Schroeder and Ristic, 1965). The importance of the spleen in resistance to infectious anemia was clearly indicated in Haemobartonella muris infections of rats by Nelson (1970). Her rats, obtained from a commercial vendor, appeared to be healthy, but when they were subjected to splenectomy every one developed patent H, meri§_infection and each died within 2 weeks of hemolytic anemia. When H, mgri§_free rats were infected they developed patent infections and in 6-8 days, they developed moderately severe anemia from which they appeared to have recovered after 4 weeks. When these rats were given superimposed infections they again had patent infections with anemia but recovered as before. Splenectomy of these recovered rats invariably led to recrudescence of the infection and hemolytic anemia with death within 2 weeks. It was therefore clear that rats did not develop classic acquired immunity that protected them from repeated H, mgri§_infection, and that such protection as did result from recovery from H, mgri§_infection was totally dependent upon the presence of the spleen. As indicated, agents of infectious anemia appear to be unable to stimulate classic acquired immunity during infection, and for this reason such agents have been referred to as compatible parasites (Cox and Iturri, 1976; Sprent, 1963). If, on the other hand, the anti- genicity of the parasite for its host could be enhanced, it should follow that classic immunity should result from infection. In experi- ments in which the mouse parasite, Plasmodium chabaudi, was adapted to laboratory rats, the infections were first introduced into splenectomized rats using infected mouse blood. These rats ultimately developed P, chabaudi parasitemia on the sixth day after infection which remained patent for only 3 days. Blood of these rats was passed to fresh splenectomized rats which developed parasitemia after 4 days and then underwent spontaneous recovery. After the fourth passage in splenectomized rats spontaneous recovery from P, chabaudi malaria was still evident. It therefore appeared that mouse P, chabaudi parasites were potent antigen for rats and remained so for many generations of parasite reproduction. It was also clear that with these parasites the absence of a spleen was no major penalty to the development of protective immunity. Intact rats recovered from this partially adapted strain were highly resistant to P, chabaudi but were no more resistant than normal rats to challenge with Babesia rodhaini. Thus in this malaria the absence of a spleen imposed no marked penalty on the development of immunity, and the resulting immunity was specific in nature rather than the nonspecific kind associated with premunition (Musoke, 1973). It therefore indicated that when the agents of infectious anemia are sufficiently antigenic for their host, classic immunity rather than premunition will result from the infection. Autoimmune Phenomena in Infectious Diseases The development of the conception that disease may have an immunologic basis has been hampered by concepts that were more emo- tional than rational in basis. In spite of the demonstration that inflammation associated with the smallpox and cowpox was clearly related to acquired immunity by Jenner (1798), the concept of an allergic basis for disease was not enunciated until 1905 when von Pirquet published his ideas in Die Serumkrankheit (von Pirquet and Schick, 1905). It is only within recent years that von Pirquet's ideas have come to have some general acceptance and physicians, the descendants of the healers of the priesthood, still largely think in terms of casting out the evil spirits that are inflicted upon man for various transgressions (Magill, 1955). 10 The concept that disease could be a self destroying process from antibodies generated against ones own body was less acceptable. The concept of Ehrlich (1900) of "horror autotoxicus" was thus ele- vated to universal law rather than being kept in the realm of a valid generalization. In dealing with the growing evidence that autoimmune disease did exist, Burnett (1959) in his much quoted "clonal selection theory" is consistent with the law of "horror autotoxicus" in the idea that animals can normally only make antibody to their antigens that are sequestered from the immune system. Thus the incongruity of a concept that allowed autoimmune disease to be a part of infectious disease remains an absurdity for the scientific mind. One may have an infectious disease or one may have an autoimmune disease. One may not have both. The skepticism over an autoimmune basis for disease remains evident in the writings of modern authorities (Glynn and Holborow, 1971). Partly through ignorance, and partly through bias, the nature of the syphilis associated Wassermann antibody has been ignored. Wassermann and associates first thought this antibody had been gen- erated by the antigens of Treponema pallidum since it was detected with a "watery extract" of liver from a syphilitic foetus which was teeming with spirochaetes (Wassermann et al., 1906; Wassermann et al., 1906). However during the following year, Marie and Lavaditi (1907) found that "watery extracts" of normal liver would also react with the antibody. It might have been suspected at that time that Wassermann's antibody was an autoantibody; however, Ehrlich (1900) was not chal- lenged. Since modern Wassermann antigen is an alcoholic extract of ll lipids from beef heart, the dissociation of Wassermann antigen from those of T, pallidum is further indicated. Portnoy and Magnuson (1955) raised both Wassermann and treponema-immobilizing antibodies by injecting extracts of T, pallidum infected rabbit testicle into normal rabbits and concluded that the antibodies were not the same, but did not speculate on the nature of the Wassermann antibody. The association of Wassermann antibody with diseases other than syphilis should have raised questions as to the nature of the antibody. Its association with yaws and pinta were accepted as valid since these were Treponema associated diseases. However, its association with other diseases was termed artifact and called "biological false positive" reactions (Wilson and Miles, 1964). Since the diseases with which "biological false positive" reactions are considerable in number, a partial listing of these diseases include malaria, kala-azar, leprosy, tuberculosis, rheumatic diseases and many others (Wilson and Miles, 1964). As indicated, Wassermann antibody is associated with a number of diseases which are mediated by a variety of unrelated infectious agents. Kahn (1951) in studying reactions of his cardiolipin antigen found that with careful adjustments of electrolyte concentration, he could detect Wassermann-like antibody in the serum of many normal humans and animals. He therefore suggested that this was a normal or universal antibody, and that when cells that contained the lipid antigens were injured in a disease process, sufficient antibody for detection by conventional methods would result. In essence, Kahn was suggesting that Wassermann antibody was autoantibody. 12 During this era of great discoveries in immunology, 1900 to 1915, a phenomenon of the aggregation of cells treated with antibody and complemented by normal bovine serum attracted the attention of Ehrlich (Ehrlich and Morgenroth, 1900; Ehrlich and Sachs, 1902a, b) and Bordet (Bordet and Gay, 1906, 1908). Bordet (Bordet and Streng, 1909) termed this aggregation conglutination to distinguish it from the true agglutination which resulted from specific antibody. He termed the factor from bovine serum "coloide de boeuf" or "conglutinin" to differentiate it from agglutinating antibody. A colleague of Bordet, Streng (1909a, b) and Streng and Ryti (1923) proved that conglutinin was a nonantibody fraction of bovine globulin that reacted with complement that was bound to an antigen-antibody complex, but that it did not react with unactivated complement. In 1930 Streng found that a conglutinin-like factor could be stimulated in rabbits, which do not have natural conglutinin, by injecting them with complement adsorbed to blood cells or to bacteria, and called this antibody immunoconglutinin (Streng, 1930). Based on the fact that immuno- conglutinin was stimulated by injections of fresh autologous serum absorbed on kaolin particles, the fact that its appearance was associ- ated with complement depletion and its specific reactivity with fixed complement, Coombs (1959) and Commbs et a1. (1961) suggested that it fulfilled the criteria for an autoantibody and that it should be associated with any event resulting in an intravascular antigen- antibody reaction. Thus immunoconglutinin should be raised by artificial stimulation, as well as infections with many infectious diseases. This appeared to be true since it has been associated with ‘1". [I'll I’llllil l [l f [. Ill {11‘ fl. 1 13 antigenic stimuli (Ingram, 1962a, b) and many diseases mediated by a variety of unrelated agents such as in acute and chronic bacterial infections (Ingram, 1965a, b), in trypanosomiasis, typhoid fever, leishmaniasis, malaria and others (Woodruff, 1973; Woodruff et al., 1972). The immunochemical and biological aspects of immunoconglutinins, of both human and rabbit origin, have been widely investigated. The great majority of the autostimulated immunoconglutinins are IgM anti- bodies (Bienenstock and Block, 1966; Lachmann, 1967). Immunocon- glutinins that are detected by direct agglutination of complement- fixed cells are generally IgM antibodies, although immunoconglutinins of IgG, IgM and occasionally IgA classes have also been detected by specific antiglobulin technique (Henson, 1968). Lachmann and Thompson (1970) reported that human saliva regularly contains quite high levels of immunoconglutinin, this being antigenically IgA. Studies of the specificity of immunoconglutinins have been made, they reacted specifically against the bound C3 (Lachmann, 1962; Lachmann and Coombs, 1965; Lachmann and Muller—Eberhard, 1968) and bound C4 (Lachmann, 1966) components of complement. Mittal and Ingram (1969) showed that natural antibodies and immunoconglutinin, in the presence of complement, acted synergistically to increase bactericidal activity. Parappally and Ingram (1973) demonstrated that immunoconglutinin enhanced phagocytic activity of mouse peritoneal macrophages against bacterial cells ip_ 31332, The amplifying effect of immunoconglutinin on complement fixation ig.vivo was shown by Tedesco and coworkers, suggesting that 14 this autoantibody potentiated the ability of complement to bring about red cell destruction (Tedesco et al., 1972). Thomsen and Friedenreich in 1928 observed that erythrocytes that had been treated with bacterial extracts would detect hemag- glutinins that could not be detected with normal blood cells (Frieden- reich, 1928). Morton and Pickles (1947) and Wiener and Klatz (1951) found that Type "0" Ph-positive erythrocytes would be agglutinated by serum from persons suffering Rh disease. The belief that these workers were detecting incomplete antibody to Rh antigen was somewhat shaken with the finding that hemagglutinin was present in sera of persons with a variety of acquired hemolytic anemias (Dacie, 1962). Dacie (1962) suggested that this hemagglutinin was a naturally occurring autoantibody and functioned normally in the removal of effete blood cells from the circulation. In hemolytic diseases antigen was exposed after cells were damaged and the titers of the antibody would reach detectable levels. This hemagglutinin was found associated with the anemia of babesiosis, anaplasmosis and malaria (Cox et al., 1966; Schroeder et al., 1966; Schroeder and Ristic, 1965) and was subse- quently found in the blood of animals suffering anemia from a variety of unrelated infectious diseases (Cox and Iturri, 1976). Soni and Cox (1975a) caused the hemagglutinin to be produced in normal chickens by injecting them with autologous trypsinized erythrocytes. Since anemia accompanying the appearance of the antibody, they concluded that it was an autoantibody and that in chickens, it was an anemia inducing factor. 15 In studying bone marrow of patients with systemic lupus erythematosus Hargreaves in 1948 (as cited by Humphrey and White, 1970) noted that many of the granulocytic cells had deformed nuclei and that vaguely outlined discs, that gave a positive Feulgen stain reaction, were frequently associated with the disease. It was subsequently found that these lupus erythematosus (LE) cells could be induced by treating normal polymorphonuclear cells with the serum of patients with systemic lupus erythematosus (SLE), and the inducing factor of the serum was identified as antibody of the IgG class (Holman, 1965). It appears that this autoantibody, termed lupus erythematosus (LE) factor, or anti-nuclear factor may consist of antibody to deosyribo- nucleic acid and to soluble extractable nuclear antigen of the cells. Stimulation of LE factor with purified fractions has not been achieved, except when they were injected as mixtures of complete Freund's adjuvent, which in itself may stimulate the antibody (Holman, 1972; Holman and Deicher, 1959). In addition to SLE, LE factor was associ- ated with New Zealand Black mouse disease and with Aleutian Mink Disease, both of which are associated with congenital viral infections (Helyer and Howie, 1963; Holman, 1972; Howie and Helyer, 1968; Mellors and Huang, 1966). SLE had been reported as a rare and late developing sequelia of infection with BCG vaccine and the Vole Mycobacterium used as prophylaxis for tuberculosis (Wilson and Miles, 1964). About 40% of rheumatoid arthritis cases have positive tests for LE factor (Holman, 1972). A more ubiquitous autoantibody is recognized in the so called rheumatoid arthritis (RA) factor associated with rheumatic and other 16 chronic diseases, both idiopathic and infectious (Christian, 1971; Humphrey and White, 1970). The discovery of RA factor was based on the observation that sera of some persons, especially those with rheumatoid arthritis, would aggregate sheep erythrocytes treated minimally with anti-sheep red blood cell serum. It was subsequently found that semi-purified IgG absorbed to particles such as tanned cells, latex or bentonite particles would be aggregated in the same manner by these sera. Recovery of the factor from complexes of it and IgG coated particles indicated that it was usually globulin of the IgG class, but was often a mixture of IgM and IgG at a ratio of one molecule of IgM to S of IgG with a sedimentation coefficient of 225. This mixture has been considered possibly as a complex of 198 antibody with 78 antigen (Metzger, 1967; Normansell and Stanworth, 1968). RA antibody appears to react with the Fc part of the IgG chain, which may in turn have been made reactive by the binding of the globulin to antigen or other particles (Milgrom and Witebsky, 1960). Thus in nature the factor may be stimulated by soluble complexes of IgG with antigen. The ability to generate this autoantibody may be under genetic control as was indicated by positive reactions within families, with or without signs of rheumatic diseases. As with other autoimmune factors, RA factor has been associated with diseases associated with a variety of unrelated infectious agents such as malaria, kala azar, syphilis, tuberculosis, leprosy and bacterial endocarditis (Barrett-Connor, 1967; Christian, 1971). Eaton (1939) found that serum of monkeys with acute Plasmodium knowlesi malaria would react with serum of persons recovered from 17 vivax, falciparum or quartan malaria. The presence of the antigen in the serum of malarious monkeys was confirmed by Cox (1966) who also reported that injections of the globulin of the malarious monkeys into rats produced an anemia and afterwards these rats were resistant to Plasmodium berghei malaria. Sibinovic et a1. (1967, 1969, 1967) detected antigen in the serum of dogs, horses and rodents with babesiosis that reacted with serum of rats recovered from Babesia rodhaini infection, dogs recovered from Babesia canis infection, and horses recovered from Babesia equi infection. Injection of globulin from dogs or rats with babesiosis caused anemia in the recipient animals and they were later resistant to infections with heterologous parasites. Cox et al. (1968) showed that the soluble antigens of serum from animals with acute malaria and babesiosis appeared to be identical in immunodiffusion tests, and Corwin and Cox (1969) found that immunization with serum containing this antigen would confer resistance to heterologous genera and species of haemosporidian para- sites. Soni and Cox (1974, 1975a, b, c) confirmed the identity of the antigens associated with malaria and babesiosis, found these anti- gens not to react with antibody to purified parasite antigen and found that the antigen as a complex with its antibody were mediators of anemia and nephritis. In addition to malaria and babesiosis, this soluble serum antigen was found associated with Eperythrozoon coccoides infections of mice and Haemobartonella muris infections of rats (Cox and Iturri, 1976). Thus this soluble antigen of serum is rather ubiquitous in its association with infectious diseases and for this reason it was suggested that it might be an autoantigen (Cox and 18 Iturri, 1976). Antibody to this antigen might be another of the auto- antibodies that are found associated with infectious diseases. Antigen and Antibody_Complexes in Diseases The first observations on disease resulting from antigen and antibody alone were those recorded by von Pirquet and Schick (1905) in the monograph, "Die Serumkrankheit." From this it was clear that you Pirquet recognized that serum sickness was a result of antibody that' had been stimulated by injections of horse serum and that the disease mimicked infectious diseases, both acute and chronic. Serum sickness became a model for the study of immunopathology, first as a local reaction in skin to study the effects on tissue of an immune animal of injected antigen as in the work of Arthus (1903, 1906), and then as a model for diseases resembling rheumatoid arthritis, lupus erythematosus, glomerulonephritis or polyarteritis (Dixon, 1971; Dixon et al., 1961; Udaka, 1971). It was established that the disease was associated with an antigen excess over antibody, which might best be achieved by intra- vascular inoculation of the antigen into nonimmune animals and watch- ing the disease develop with the appearance of antibody, however similar results were achieved by the intravenous injection of non- precipitating mixtures of antigen and antibody with antigen in excess (Germuth et al., 1967). The demonstration that immune complexes could be induced by the injection of autoantigens and that the appearance of these complexes was accompanied by signs of glomerulonephritis indicated that immune complex disease might be a functional mechanism in certain autoimmune diseases (Edgington et al., 1967). Experimental 19 serum sickness has become a popular model for the study of the immunopathology of vascular-renal disease, and the role of complexes and complement fragments activated by complexes, in arteritis and glomerulonephritis have been well studied by McCluskey and Vassalli (1971) and Dixon (1971). An additional aspect of immune complex disease has received less attention. Dixon (1966) suggested that soluble antigen-antibody complexes could bind nonspecifically with blood cells or other particles in blood, usually by means of the Fc chain of the antibody (Dixon et al., 1961). With the bound glubulin of the complex, the cells and particles will act as though they were opsoninized and will be sequestered in the spleen, or if conditions are correct, the cells or particles may activate complement and undergo intravascular lysis. Soni and Cox (1974, 1975b) verified this suggestion of Dixon (1966) by finding that injection of malarious globulin containing soluble serum antigen and its antibody into normal birds immediately resulted in anemia and that the antigen moiety of the complex could be detected on the surface of blood cells of the recipient birds by fluorescent antibody methods. Thus antigen-antibody complexes may be anemia inducing factors as well as the mediators of arteritis or glomeru- lonephritis. An unexplored ramification of antigen-antibody complex activity is their role as determinants for autoimmunization. Immunocon- glutinins are autoantibodies stimulated by altered C and C4 fragments 3 of complement (Lachmann, 1962, 1966; Lachmann and Coombs, 1965). Similarly, rheumatoid arthritis factor is autoantibody to the Fc 20 region of IgG that has been modified in some way when it forms a complex with antigen (Milgrom and Witebsky, 1960). It may be possible that complexes nonspecifically bound to erythrocytes may alter the cell membrane to the extent that the stroma antigens of Thomson and Friedenreich (Friedenreich, 1928) are expressed and therefore indirectly be responsible for the stimulation of autohemagglutinins. Similarly, the interaction of complexes with vascular endothelium or other cells may damage the cells to release the antigenic lipid that stimulate Wassermann antibody as was suggested by Kahn (1951). The fact that these autoantibodies might in turn form immune complexes with their antigens has received little literary consideration. The Agents of Infectious Anemia The agents of infectious anemia form a taxonomically diverse group which may be parasites of an almost equally diverse group of hosts, e.g., man, equines, canines, ovines, bovines, rodents, and avians. Thus viral infectious anemia has been a rec0gnized disease in horses, ducks, and more recently, in laboratory rats. Agents of the rickettsia-like agents, Bartonella, Anaplasma, Haemobartonella, Eperythrozoon and perhaps others, may produce infectious anemia in man, swine, sheep, cattle, dogs, cats and rodents. Protozoan para- sites of the Plasmodium, Babesia, and Trypanosoma groups are well known associates of the infectious anemia syndrome in man, various domestic animals and birds. Infections of agents listed have been associated with anemia and splenomegaly which were accompanied by a common autoimmune factor, cold-active hemagglutinin (Cox and Iturri, 21 1976; Cox et al., 1966; Oki and Miura, 1970; Schroeder et al., 1966; Schroeder and Ristic, 1965). While the list of agents is incomplete, it clearly illustrates their taxonomic diversity. The infectious anemia syndrome may be associated with other infectious diseases, but with less frequency and in less dramatic ways. Anemia with splenomegaly and nephritis accom- panied by autoimmune factors is commonly associated with Kala Azar, a generally fatal chronic disease which may result from infections of Leishmania donovani (Woodruff, 1973; Woodruff et al., 1972). In some cases of advanced syphilis hemoglobinuric anemia will be accompanied by the presence of autohemagglutinins (Donath and Landsteiner, 1904). Signs of jaundice or red water disease may be seen with considerable frequency in leptospirosis of man and animals (Wilson and Miles, 1964). During the acute viremic stages of Cytomegalovirus or Epstein-Barr virus, anemia with splenomegaly has been noted with some frequency (Jawetz et al., 1974). J. L. Soni (personal communication) observed anemia with splenomegaly and nephritis accompanied by cold-active hemagglutinin in chickens during an outbreak of Borrelia anserina infection at the Veterinary College at Jabalpur, India. Thus the number and diversity of agents that have been associated with the infectious anemia syndrome may be considerably greater than was indicated. 0n the other hand the syndrome may be inconspicuous during the infections of the agents listed. Marchiafavia (1931) commented on the frequency with which he had seen patients whose blood was teeming with Plasmodium falciparum parasites who had not and did not later have 22 symptoms relatable to pernicious malaria. Martin Young (personal communication) tells of calling Liberian or Panamanian children from their play to prick their fingers and then to find that the blood of these same children was teeming with parasites. Hackett (1937) pro- tested that the Italians he found heavily infected with plasmodial parasites had few complaints that were relatable to malarial disease. J. L. Soni (personal communication) told of the frequency with which he had seen heavy infections of Babesia bigemina in Indian cattle that had no signs of the red water disease, which was associated with infection with this parasite during the Texas epizootic described by Smith and Kilborne (1893). Thus the absence of the infectious anemia syndrome during acute infection may be as much of a phenomenon as is its presence. Agents of Infectious Anemia Used for Comparative Study The objectives of the present research are to determine the nature of autoimmune-like factors which may be related to the patho- genesis and to the phenomenon of nonspecific acquired resistance in infectious anemias. It was therefore desirable that agents of taxonomic diversity which consistently induced the syndrome in a single host species be used for the study. As a host, Sprague-Dawley male rats were selected since much of the earlier works relating to anemia and nephritis from malarial, babesial and Haemobartonella infections had employed laboratory rats (Cox, 1966; Cox and Iturri, 1976; Cox et al., 1969; Schroeder et al., 1966; Sibinovic et al., 1967). 23 Two fortunate observations made from this laboratory provided two unrelated agents for the study. It was observed that a laboratory strain of Trypanosoma lewisi maintained at this laboratory produced unusually severe anemia during infections of rats. The trypanosome infection was eliminated by infecting rats that had been made immune by infection with a new strain of I, lewisi obtained from the American Type Culture Collection which is here designated as the ATC strain. These rats did not show trypanosome parasites in their blood, but on the seventh day after infection they showed signs of hemolytic anemia. Blood was transferred from these to fresh rats which in turn did not develop trypanosomal parasites, but deve10ped hemolytic anemia and died within 8 days. This trypanosome free agent has been maintained by blood passage in rats, or as frozen infected blood in liquid nitrogen since June 1974. The agent passed a 0.2 micron filter and could not be cultured on media for bacteria, leptospira or mycoplasma. No organisms were detected by microscopic examination of blood of infected animals. The agent was not infective when inoculated into mature mice, and rats housed in the cage with infected rats did not become infected. While evidence is not complete, it is believed that this filterable replicating agent is a virus and that it can be handled with safety as a laboratory infection. In rats the agent causes hemolytic anemia, splenomegaly and acute glomerulonephritis which are accompanied by the presence of high titers of cold-active hemagglutinin (CAH). Mature rats usually die of the disease within 8 to 10 days after inoculation. Young rats weighing 100 gm or less invariably recover and appear normal 30 days after 24 infection. Reinfection of recovered rats results in reappearance of anemia from which the rats would recover. Studies of the scientific literature has not furnished information on an anemia inducing viral agent that is exclusively a parasite of rats. Since it fulfills the characteristics for infectious anemia, the disease has been referred to as rat infectious anemia (RIA). Details of the isolation of RIA virus and a description of the disease is presented elsewhere in this thesis (Thoongsuwan and Cox, 1976a). During the isolation of RIA virus from its trypanosome com- panion, it was noted that rats infected with the new ATC strain of I, lewisi developed moderately severe anemia with splenomegaly, from which they did not completely recover until as late as 30 days after infection. An investigation of this infection was initiated since I, lewisi had been characterized as nonpathogenic for rats (Kudo, 1947; Soulsby, 1968). The study indicated that there was anemia with splenomegaly and mild glomerulonephritis accompanied by the presence of cold-active hemagglutinin (CAH) associated with the post-acute phase of I: lewisi infection of rats. The details of this study are reported elsewhere in this thesis (Thoongsuwan and Cox, 1976b). These preliminary investigations have furnished two taxonomically unrelated agents, a filterable replicating agent presumed to be virus, and a hemoflagellate, I, lewisi, which may produce the infectious anemia syndrome in laboratory rats. For comparative purposes two haemosporidian parasites, Plasmodium chabaudi and Babesia rodhaini, were selected since previous study had indicated that the infectious anemia syndrome was associated with the infection of each in laboratory 25 rats (Iturri and Cox, 1969; Musoke, 1973; Schroeder et al., 1966; Thoongsuwan, 1971; Thoongsuwan and Cox, 1973). History and Taxonomy of the Agents Studied Filterable Rat Infectious Anemia (RIA) Agent As indicated, previous records of a viral agent of rats that caused disease signs resembling those of RIA were not found. However there was a single account of anemia resulting from the infection of newborn rats with Friends Murine Leukemia virus (Kuzumaki et al., 1974). In this work it was reported that the rats developed signs of leukemia and lymphomas in addition to anemia. Evidence of leukemia or tumors in rats infected with RIA agent was not found, and since RIA agent appeared not to be infective for mice, it was considered improbable that it could have been Friends virus (Thoongsuwan and Cox, 1976a). It was therefore assumed that RIA agent and RIA disease had not been previously described. The other agents employed were protozoan parasites that had been known for some time and had been adequately classified by Levine (1961) whose taxonomic terminology will be followed. Trypanosoma lewisi Trypanosoma lewisi is believed to be the first trypanosome parasite found in mammalian blood (Levine, 1961). In classification it and the other trypanosomes are grouped in Class Mastigasida, the protozoans without pseudopodia, with one or more flagella and a vesicular nucleus. It is included along with those having one or two 26 flagella in Order Promastigorida and with those with a single flagellum, a single nucleus, a kinetoplast and a parabasal body which given rise to the flagellum, in Family Trypanosomatidae. The genera of the family are exclusively parasitic, and in addition to Genus Trypanosoma, include other important parasites of vertebrates, Genus Leishmania. Other genera of the family, which are primarily parasites of arthropods and plants, must be mentioned since the trypanosomes may resemble them in form during the various stages of development in their life cycles (Levine, 1961). Arthropod parasites of Genus Leptomonas may alternate between two forms: the first, a leptomonad stage, having a parabasal body located at the anterior pole of the cell and the flagellum attached to it emerging anteriorly without attachment to the ectoplasm by an undulating membrane, and secondly an unflagellated oval intracellular form resembling leishmanial parasites. ArthrOpod parasites of Genus Crithidia have a single form resembling the leptomonad except that the parabasal body is more centrally located and the flagellum emerges laterally and is attached to the ectoplasm as it proceeds anteriorally. In their cyclic development, trypanosomes may have leptomonal, crithidial, leishmanial or trypanosomal stages. In the latter form the parabasal body is posteriorally and the emerging flagellum is attached to the ectoplasm from the posterior to the anterior end by means of an undulating membrane (Kudo, 1947). This descriptive nomen- clature appears to have caused confusion and modern texts refer to leishmanial forms as amastigotes, leptomonal forms as promastigotes, 27 crithidial forms as epimastigotes and trypanosomal forms as trypo- mastigotes (Faust et al., 1975). Among the trypanosomes in general, the trypanosomal forms are found as actively motile extracellular parasites of blood and the leishmanial forms are found as intracellular parasites of monocytes or the phagocytic histiocytes of various tissues. Leptomonal and crithidial stages are found as extracellular parasites of the foregut, the hindgut or the salivary glands of their arthropod vector hosts. The crithidial form of trypanosomes, which is infective for the verte- brate host, will be located in the mouth parts or salivary glands of arthropods infected with certain trypanosomes. Crithidial forms of others will be found in the hindgut and will be passed from the vector host in its feces. Thus parasites of some trypanosomes are passed to a new host by the bite of the blood sucking arthropod while others are passed by the contaminated feces being rubbed into wounds made by the feeding arthropod or by contamination of oral mucosa or the con- juctiva (Faust et al., 1975). Other trypanosomes, perhaps as a result of adaptation, appear to have lost their biological requirement for an arthropod host. Trypanosoma evansi, the agent for surra in equines or camels, is passed mechanically by the contaminated proboscis of blood feeding biting flies such as Tabanus spp. or Stomoxys spp., Trypanosoma eqeiperdum, the agent of equine dourine or equine syphilis, is usually transmitted venerally (Levine, 1961). Historically, the first trypanosome was described from the blood of salmon by Valentin in 1841 and from the blood of a frog by 28 Gluge in 1842. Lewis in 1878 described the first trypanosome from mammals in the blood of rats and Evans in 1881 was the first to relate trypanosomes to disease. Bruce in 1895 discovered the tsetse fly (Genus Glossina) as a host for African trypanosomes and in 1902 Dutton related Trypanosoma gambiense to the human disease, African sleeping sickness (Levine, 1961). I, lewisi is distributed worldwide as a parasite of the common black rat, Rattus rattus, and the more common Norway rat, Rattus norvegicus, in which it appeared not to produce disease. It was readily adapted by blood passage to laboratory rats and has been referred to as nonpathogenic (Kudo, 1947; Soulsby, 1968). Its bio- logical vector host, the rat flea Ceratophyllus fasciatus, becomes infected while taking blood from the infected rat. In the stomach of the flea, the parasite infects epithelial cells of the organ and multiplies. The emerging crithidial forms migrate to the hindgut where they may pass in the feces of the flea or may infect the epi- thelium of the gut. In feeding, fleas move freely from host to host and they defecate profusely. Thus the rat in grooming, or scratching could readily acquire infection from this ectOparasite (Kudo, 1947). Since it is easily handled in the laboratory and is a potent immunogen for rats, I, legiei has been used as a model for the study of trypanosomal immunity since as early as 1899 (Rabinowitsch and Kempner, 1899). The ATC strain used for this research was given to the American Type Culture Collection by Dr. Robert G. Yaeger of Tulane University School of Medicine, New Orleans, La. In observation of 29 this parasite at this laboratory no evidence of RIA virus contamination has been found. The haemosporiaian parasites, Plasmodium chabaudi and Babesia rodhaini, have been described in previous reviews (Musoke, 1973; Thoongsuwan, 1971). Plasmodium chabaudi The P, chabaudi strain was adapted from laboratory mice to rats by Musoke (1973). The strain had been used in previous investi- gations from this laboratory (Cox and Milar, 1968; Cox et al., 1968). It was provided by the late Dr. Elvio H. Sadun, then Director of the Department of Medical Zoology, Walter Reed Army Institute of Research, Washington, D.C., and was cleared of Eperythrozoon coccoides contami- nation by methods recommended by Thompson and Bayles (1966). When first adapted, this strain appeared to be a potent antigen and even splenectomized rats spontaneously recovered. With passages, the antigenicity appeared to have diminished, and after the 35th passage, all infected rats died during a hemolytic crisis (Musoke, 1973). P, chabaudi was isolated from the blood of a tree rat, Thallomys rutilans, of the Central African Republic by Landau (1965), who was unable to find the natural vector mosquito (Landau and Chabaud, 1965). When it was transferred to laboratory mice E, coccoides con— tamination may have contributed to a false characteristic in that it was considered less lethal for mice than the previously described Plasmodium berghei and Plasmodium vinckei, which were also parasites of Central African tree rats (Ott et al., 1967). Elimination of the E, coccoides left P, chabaudi as lethal for mice as were the other I III i.i i l {l [I [[1 [[ (III. ..III‘ [(Illl l.‘ I lit {(III. Ii' il‘ 30 parasites, and recontamination resulted in mice being spared the lethal effects (Ott et al., 1967). Like P, vinckei, P, chabaudi infects mature erythrocytes with much greater frequency than does 2, berghei, which preferentially infects reticulocytes, this characteristic remained evident after P, chabaudi had been adapted to rats (Musoke, 1973). Malaria, the disease of human plasmodial infections, was described as a recognizable entity in Ebers Papyrus dating from 1500 B.C. The disease was related to bad air and mosquitoes in the Edwin Smith Surgical Papyrus dating from 1600 B.C. (Breasted, 1930; Halawani and Shawarby, 1957). Specific curative and suppressive drug, Quinine, was found in common use by South American Indians and was introduced as an antimalarial drug to Europe in 1640 (de Angelis, 1954). The etiologic agents for the malarial fever were not discovered until they were described in 1878 in the blood of French soldiers in Algeria by Laveran (1880, 1884). Remarkably, these observations of the three common species of human plasmodia were made without the aid of the oil immersion lens and the polychrome staining methods that are so heavily relied upon today. It remained for Ross (Ross, 1897, 1898) to discover the developmental steps of plasmodial parasites in mosquitoes employ- ing parasites of the common magpies of India. The mosquito phase of human plasmodial parasites was reported at about the same time, by Grassi (1900) in Italy. Knowledge of the complete life cycle was furnished in 1944 by the observations of Huff and coworkers (Huff and Bloom, 1935; Huff and Coulston, 1944) on the exoerythrocytic schizogony cycle of the plasmodial parasites. 31 Babesia rodhaini The strain of g, rodhaini used at this laboratory was obtained from the late Dr. Paul E. Thompson who was then with Parke Davis and Company, Ann Arbor, Michigan. It was used extensively as a model infection in laboratory rats (Cox and Milar, 1968; Cox et al., 1968; Iturri and Cox, 1969; Schroeder et al., 1966; Thoongsuwan and Cox, 1973). E, rodhaini is another of the haemosporidian parasites found in tree rats of Central Africa. It was found in the blood of an arboreal rat, Thamnomys surdaster surdaster in the then Belgian Congo in 1950 and was adapted to laboratory mice by Van den Berghe and coworkers (1950). The arthropod vector was not discovered but was assumed to be an Ixodid tick. The parasite was adapted to laboratory rats by Beveridge (1953). Early taxonomists had included Genera Babesia and Plasmodium within Order Haemosporidia of Class Sporozoa, Phylum Protozoa (Kudo, 1947). Rearrangement of the sporulating protozoans into new classes and subclasses as recommended by Levine (1961) has not contributed to clarity and some of the newer textbooks retain the older arrangement (Kudo, 1947; Brown, 1975). Since the life cycles and taxonomic rela- tionships of a number of these parasites have not been elucidated, it is here preferred to leave the babesial and plasmodial parasites in the Order Haemosporidia. Babesial parasites were first observed in the blood of African cattle suffering red water disease in 1888 by Babes (1888), and the parasites were given the name Babesia in his honor in 1893 by 32 Starcovici (1893). Hard ticks were identified as the arthropod vector by Smith and Kilbourne (1893) while they were working on the great outbreak of the disease in Texas. They also demonstrated that the parasites were passed from adult to young ticks via infected eggs. The developmental cycle of Babesia parasites in Ixodid ticks has not been completely elucidated. Babesiosis is recognized as an important disease of many domestic and wild animals. It is of passing interest that the pet lioness of Mrs. Joy Adamson of Kenya, Elsa of her book, "Born Free," was reported to have died of babesiosis. Dr. C. G. D. Brown of Kenya has estimated that babesiosis, along with trypanosomiasis and East Coast Fever, caused by the tick borne Theilaria parva, may have killed as many as three million head of cattle in East Africa within a single year (personal communication). The recent discovery of several cases of human babesiosis has raised a concern that these parasites might also be a human health hazard in parts of the world (Anderson et al., 1974). REFERENCES REFERENCES Anderson, A. E., P. B. Cassady, and G. R. Healy. 1974. Babesiosis in man, sixth documented case. Am. J. Clin. Path. 62:612-618. Arthus, M. 1903. Injections répétéis de serum de cheval chez le lapin. C. R. Soc. Biol. 55:817. Arthus, M. 1906. Sur la séro-anaphylaxic de lapin. C. R. Soc. Biol. 60:1143. Babes, V. 1888. Sur 1'hemoglobinuria bacterienne de boeufs. Compt. Rend. Acad. Sci. 107:692-700. Barrett-Conner, E. 1967. Plasmodigm vivax malaria and Coombs- positive anemia. Amer. J. Trop. Med. Hyg. 16:699-703. Beveridge, E. 1953. Babesia rodhaini: a useful organism for the testing of drugs designed for the treatment of piroplasmosis. Ann. Trop. Med. Parasitol. 47:134-138. Bienenstock, J. and K. J. Bloch. 1966. Some characteristics of human-immunoconglutinin. J. Immunol. 96:637-645. Bordet, J. and F. P. Gay. 1906. Sur les relations des sensibilisatrices avic 1' alexine. Ann. Inst. Pasteur. 20:467-498. Bordet, J. and F. P. Gay. 1908. L' absorbtion de 1' alexine et le pouvoir antagoniste des sérums normaux. Ann. Inst. Pasteur. 22:625-643. Bordet, J. and O. Streng. 1909. Les phénomenes d' absorbtion et la conglutinine du serum de boeuf. Zbl. Bakt. (Orig.) 49: 206-276. Breasted, J. H. 1930. The Edwin Smith surgical papyrus. University of Chicago Press, Chicago, Illinois. Brown, H. W. 1975. Basic clinical parasitology, 4th Ed. Appleton- Century-Crofts, New York. 33 34 Burnett, F. M. 1959. The clonal selection theory of acquired immunity. Cambridge University Press. Christian, C. L. 1971. Rheumatoid arthritis. In: Immunological Coombs, Coombs, Corwin, Cox, F. Cox, H. Cox, H. Cox, H. Cox, H. Cox, H. Cox, H. Cox, H. Diseases, 2nd Ed., M. Samter (Ed.), Vol._2; pp. 1014-1028. Little, Brown and Company, Boston. R. R. A. 1959. Concepts and mechanisms of pathogenic immuno-reactions. IE} Immunopathology, lst International Symposium, P. Grabar and P. Miescher (Eds.), Benno Schwabe, Basel, pp. 13-19. R. R. A., A. M. Coombs, and D. G. Ingram. 1961. The serology of conglutination and its relation to disease. Charles C. Thomas, Publisher, Springfield, Illinois. R. M. and H. W. Cox. 1969. The immunogenic activities of the nonspecific serum antigens of acute haemosporidian infections. Mil. Med. (Suppl.) 134:1258-1265. E. G. 1972. Protective heterologous immunity between Plasmodium atheruri and other Plasmodium spp. and Babesia spp. in mice. Parasitol. 65:379-387. W. 1964. Comments on autoimmunity in malaria. Amer. J. Trop. Med. Hyg. 13:225-227. W. 1966. A factor associated with anemia and immunity in Plasmodium knowlesi infections. Mil. Med. (Suppl.) 131: 1195-1200. W. 1969. The effect of antisera to normal rat and mouse erythrocytes on the infectivity of Plasmodium berghei. Mil. Med. (Suppl.) 134:1266-1275. W. and G. Calaf-Iturri. 1976. Autoimmune factors associated with anemia in acute HaemobarEenella and Eperythrozoan infections of rodents. Ann. Trop. Med. Parasitol. 70:73-79. W. and R. Milar. 1968. Cross-protection immunization by Plasmodium and Babesia infections of rats and mice. Amer. J. Trop. Med. Hyg. 17:173-179. W., R. Milar, and S. Patterson. 1968. Serologic cross- reactions of serum antigens associated with acute Plasmodium and Babesia infections. Amer. J. Trop. Med. Hyg. 17:15-18. W., W. F. Schroeder, and M. Ristic. 1966. Hemagglutination and erythrophagocytosis associated with the anemia of Plasmodium berghei infections of rats. J. Protozool. 13: 327-332. 35 Dacie, J. V. 1962. The hemolytic anemias, congenital and acquired. Vol. II. Autoimmune hemolytic anemias. J. A. Churchill Ltd., London. de Angelis, P. 1954. Le_spezieria dell' Arcispedole ge_Santo Spirito e_le_lotta contro le_malaria. Collana di Studi storici sull' ospedali di S. Spirito in Saxia, Rome. Dixon, F. J. 1966. Comments on immunopathology. Mil. Med. (Suppl.) 131:1233-1234. Dixon, F. J. 1971. Experimental serum sickness. lg; Immunological Diseases, 2nd Ed., M. Samter (Ed.), Vol. I, pp. 253-264. Little, Brown and Company, Boston. Dixon, F. J., J. D. Feldman, and J. J. Vasquez. 1961. Experimental glomerulonephritis: The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis. J. Exp. Med. 113:899-920. Donath, J. and K. Landsteiner. 1904. Ueber paroxysmale hfimoglobinurie. Munchen Med. Wschr. 51:1590. Eaton, M. D. 1939. The soluble malarial antigen in the serum of monkeys infected with Plasmodium knowlesi. J. Exp. Med. 69:517-532. Edgington, T. S., R. J. Glassock, and F. J. Dixon. 1967. Autologous immune-complex pathogenesis of experimental allergic glomerulonephritis. Science 155:1432. Ehrlich, P. 1900. On immunity with special reference to cell life. Proc. Roy. Soc. London 66:424. Ehrlich, P. and J. Morgenroth. 1900. Ueber hamolysine. Berl. Klin. Wschr. 37:681-687. Ehrlich, P. and H. Sachs. 1902a. Ueber die visheit der complemente des serums. Berl. Klin. Wschr. 39:296-299, 335-338. Ehrlich, P. and H. Sachs. 1902b. Ueber den mechanismus der ambocestorenwirking. Berl. Klin. Wschr. 39:492-496. Faust, E. C., P. C. Beaver, and R. C. Jung. 1975. Animal agents and vectors of human disease. 4th Ed., Lea G Febiger, Philadelphia. Fosdick, W. M., J. L. Parsons, and D. F. Hill. 1968. Long-term cyclophosphamide therapy in rheumatoid arthritis. Arthritis Rheum. 11:151. Friedenreich, V. 1928. Investigation into the Thomsen hemagglutination phenomenon. Acta. Path. Microbiol. (Scand.) 5:59-101. 36 Germuth, F. 6., Jr., L. B. Senterfit, and A. D. Pollack. 1967. Immune complex disease. 1. Experimental acute and chronic glomerulonephritis. Johns Hopkins Med. J. 120:225. Glynn, L. E. and E. J. Holborow. 1971. Mechanisms of autoaggression. IE} Inflammation, Immunity and Hypersensitivity, H. C. Movat (Ed.), pp. 298-332. Harper and Rowe, Publishers, New York. Grassi, B. 1900. Studi di un zoologo sulla malaria. Rome. Greenwood, B. M. and A. M. Greenwood. 1971. Malaria infection in adult NZB mice, and in adult (NZB x NZW) F1 mice. Trans. Roy. Soc. Trop. Med. Hyg. 65:581-585. Greenwood, B. M., E. M. Herrick, and A. Voller. 1970. Suppression of autoimmune disease in N28 and (NZB x NZW) F hybrid mice by infection with malaria (Correspondence). N ture (London) 226:267. ‘Greenwood, B. M. and A. Voller. 1970a. Suppression of autoimmune disease in New Zealand mice associated with infection with malaria. I. In (NZB x NZW) F1 hybrid mice. Clin. Exp. Immunol. 7:793-803. Greenwood, B. M. and A. Voller. 1970b. Suppression of autoimmune disease in New Zealand mice associated with infection with malaria. II. In NZB mice. Clin. Exp. Immunol. 7:805-815. Hackett, L. W. 1937. Malaria in Europe. Oxford University Press, London. Halawani, A. and A. A. Shawarby. 1957. Malaria in Egypt. J. Egypt. Med. Assoc. 40:753-792. Helyer, B. J. and J. B. Howie. 1963. Renal disease associated with positive lupus erythematosus tests in a crossbred strain of mice. Nature (London) 197:197. Henson, J. B., J. R. Gorham, R. B. Leader, and B. M. Wagner. 1962. Experimental hypergammaglobulinemia in mink. J. Exp. Med. 116:357-364. Henson, P. M. 1968. Immunoconglutinins of different immunoglobulin classes demonstrated by the antiglobulin reaction. Immunol. 14:697-705. Heymann, W., D. B. Hackel, L. Harwood, S. G. F. Wilson, and J. L. P. Hunter. 1959. Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions. Proc. Soc. Exp. Biol. Med. 100:660. 37 Holman, H. R. 1960. The LE cell phenomenon. Ann. Rev. Med. 11:231. Holman, H. R. 1965. Partial purification and characterization of an extractable nuclear antigen which reacts with SLE sera. Ann. N. Y. Acad. Sci. 124:800. Holman, H. R. 1972. Systemic lupus erythematosus. 12; Immunological Diseases, 2nd Ed., Vol. 2, M. Samter (Ed.), pp. 995-1013. Little, Brown and Company, Boston. Holman, H. R. and H. R. Deicher. 1959. The reaction of the lupus erythematosus (LE) cells factor with deoxyribonucleoprotein of the cell nucleus. J. Clin. Invest. 38:2059. Howie, J. B. and B. J. Helyer. 1968. The immunology and pathology of NZB mice. Advances in Immunol. 9:215. Huff, C. G. and W. Bloom. 1935. A malarial parasite infecting all blood and blood-forming cells of birds. J. Inf. Dis. 57:315- 336. Huff, C. G. and F. Coulston. 1944. The development of Plasmodium gellinacium from sporozoite to erythrocytic trophozoite. J. Inf. Dis. 75:231-249. Humphrey, J. H. and R. G. White. 1970. Immunology for students of medicine, 3rd Ed. Blackwell Scientific Publications, Oxford and Edinburgh. Ingram, D. G. 1962a. The production of immunoconglutinin. 1. Factors affecting the response of mice to heterostimulation. Canad. J. Microbiol. 8:297-306. Ingram, D. G. 1962b. The production of immunoconglutinin. III. Factors affecting the response to auto-stimulation in mice. Canad. J. Microbiol. 8:335-344. Ingram, D. G. 1965a. The production of immunoconglutinin. IX. In acute bacterial infections. Canad. J. Microbiol. 11: 151-160. Ingram, D. G. 1965b. The production of immunoconglutinin. X. In chronic bacterial infections. Canad. J. Microbiol. 11:161- 165. Iturri, G. M. and H. W. Cox. 1969. Glomerulonephritis associated with acute haemosporidian infection. Mil. Med. (Suppl.) 134: 1195-1200. ' Jawetz, E., J. L. Melnick, and E. A. Adelberg. 1974. Review of medical microbiology. 11th Ed., pp. 442-445. Lange Medical Publications, Los Allos, California. 38 Jenner, E. 1798. An inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western countries of England, particularly Gloustershire and known by the name of the cow pox. Printed for the author by Sampson Low, Soho, London. Kahn, R. L. 1951. Universal serologic reaction in health and disease. The Commonwealth Fund, New York. Kudo, R. R. 1947. Protozoology. 3rd Ed., Charles C. Thomas Pub- lisher, Springfield, Illinois. Kuzumaki, N., T. Kodama, N. Takeichi, and H. Kobayashi. 1974. Friend lymphatic leukemia virus-induced autoimmune hemolytic anemia in rats. Int. J. Cancer 14:483-492. Lachmann, P. J. 1962. A comparison of some properties of bovine conglutinin with those of rabbit immunoconglutinin. Immunol. 5:687-705. Lachmann, P. J. 1966. A sedimentation pattern technique for measuring conglutination: Its application to demonstrating immuno- conglutinins to C4. Immunol. 11:263-271. Lachmann, P. J. 1967. Conglutinin and immunoconglutinins. Adv. Immunol. 6:479-527. Lachmann, P. J. and R. R. A. Coombs. 1965. Complement, conglutinin and immunoconglutinins. lg; Complement: Ciba Foundation Symposium (Ed. by G. E. W. Wolstenholme and J. Knight), pp. 242-273. Little, Brown and Company, Boston. Lachmann, P. J. and H. J. Muller-Eberhard. 1968. The demonstration in human serum of "conglutinogen-activating factor" and its effect on the third component of complement. J. Immunol. 100:691-698. Lachmann, P. J. and R. A. Thompson. 1970. Immunoconglutinins in human saliva--a group of unusual IgA antibodies. Immunol. 18:157-169. Landau, I. 1965. Description de Plasmodium chabaudi n.s.p., parasite de rongeurs Africaine C. R. hebd. Seanc. Acad. Sci., Paris 260:3758-3761. Landau, I. and A. G. Chabaud. 1965. Infection naturelle par deux Plasmodium du rongeur Thallomys rutilans in Republique Centre Africaine. C. R. hebd. Seanc. Acad. Sci., Paris 260:230-232. Lavaran, A. 1880. Note sur un nouveau parasite trouvé dans le sang de plusieurs malades atteints de fiévre palustre. Bull. Acad. Med., Paris, pp. 1235-1236. 39 Lavaran, A. 1884. Traité des fiévres palustres. Octave Doin., Paris. Levine, N. D. 1961. Protozoan parasites of domestic animals and of man. Burgess Publishing Company, Minneapolis, Minnesota. Ludford, C. G., R. M. Corwin, H. W. Cox, and T. A. Sheldon. 1969. Resistance of ducks to a Plasmodium sp. induced by a filterable agent. Mil. Med. (Suppl.) 134:1276-1283. Ludford, C. G., H. G. Purchase, and H. W. Cox. 1972. Duck infectious anemia virus associated with Plasmodium lophurae. Exp. Parasitol. 31:29-38. Magill, T. P. 1955. The immunologist and the evil spirits. J. Immunol. 74:1-7. Marchiafava, E. 1931. Pernicious malaria. Am. J. Hyg. 13:1-40. Marie, A. and C. Lavaditi. 1907. Ann. Inst. Pasteur. 21:138. McCluskey, R. T., F. Miller, and B. Benacerraf. 1962. Sensitization to denatured autologous gamma globulins. J. Exp. Med. 115: 253-273. McCluskey, R. T. and P. Vassalli. 1971. Serum sickness (Immune complex disease). 12; Inflammation, Immunity and Hyper- sensitivity. H. Z. Movat (Ed.), pp. 426-457. Harper and Rowe, Publishers, New York. McHardy, N. 1972. Protective effect of haemolytic serum on mice infected with Babesia rodhaini. Ann. Trop. Med. Parasitol. 66:1-5. McHardy, N. 1974. The effects of injecting anti-erythrocyte serum into calves infected with Anaplasma marginale. Ann. Trop. Med. Parasitol. 68:51-57. Mellors, R. C., T. Aoki, and R. J. Huebner. 1969. Further impli- cation of murine leukemia-like virus in the disorders of NZB mice. J. Exp. Med. 129:1045-1062. Mellors, R. C. and C. Y. Huang. 1966. Immunopathology of NZB/BL mice. V. Virus-like (filterable) agent separable from lymphoma cells and identifiable by electron microscopy. J. Exp. Med. 124:1031-1038. Metzger, H. 1967. Characterization of a human macroglobulin. V. A Waldenstrfim macroglobulin with antibody activity. Proc. Nat. Acad. Sci. 57:1490. 4o Milgrom, F. and E. Witebsky. 1960. Studies on the rheumatoid and related serum factors. 1. Autoimmunization of rabbits with gamma globulin. J. A.M.A. 174:56. Mittal, K. R. and D. G. Ingram. 1969. Bactericidal activity of rabbit serum containing immunoconglutinin. Immunol. 17: 677-684. Morton, J. A. and M. M. Pickles. 1947. Use of trypsin in the detection of incomplete anti-Rh antibodies. Nature 159:779-780. Musoke, A. J. 1973. Immunologic and pathologic studies of infections with rat-adapted Plasmodium chabaudi. M.S. thesis, Michigan State University, East Lansing, Michigan. Nelson, E. F. 1970. Preliminary studies on the disease mechanism of infectious anemia using Haemobartonella muris infections of rats. M.S. thesis, Michigan State University, East Lansing, Michigan. Normalsell, D. E. and D. R. Stanworth. 1968. Interactions between rheumatoid factor and native gamma G globulins studied in the ultracentrifuge. Immunology 15:549-560. Oki, Y. and K. Miura. 1970. Characteristics and roles of red cell antibodies in equine infectious anemia. Jap. J. Vet. Res. 32:217-227. Ott, K. J., J. K. Austin, and L. A. Stauber. 1967. Eperythrozoon coccoides and rodent malaria: Plasmodium chabaudi and Plasmodium berghei. Exp. Parasitol. 21:68-77. Parappally, N. P. and D. G. Ingram. 1973. Role of immunoconglutinin in resistance to infection: The ig_vitro promotion of phago- cytosis by immunoconglutinin. Immunol. 25:523-530. Peters, W. 1965. Competitive relationship between Eperythrozoon coccoides and Plasmodium berghei in the mouse. Exp. Parasitol. 16:158-166. Portnoy, J. and H. J. Magnuson. 1955. Immunologic studies with fractions of virulent Treponema pellidum. 1. Preparation of an antigen by desoxycholate extraction and its use in com- plement fixation. J. Immunol. 75:348-355. Rabinowitsch, L. and W. Kempner. 1899. Beitragzur kenntnissder gluparasiten, speciell der ratten trypanosomen. Ztschr. Hyg. U. Infkr. 30:251-294. Ross, R. 1897. On some peculiar pigmented cells found in two mos- quitoes fed on malarial blood. Br. Med. J. 2:1796. 41 Ross, R. 1898. Report on the cultivation of Proteosoma, Labbi, in grey mosquitoes. Govt. Press, Calcutta. Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Anaemia, para- sitaemia, erythrophagocytosis and haemagglutinins in Babesia rodhaini infection. Ann. Trop. Med. Parasitol. 60:31-38. Schroeder, W. F. and M. Ristic. 1965. Anaplasmosis. XVII. The relationship of autoimmune processes to anemia. Amer. J. Vet. Res. 26:239-245. Sergent, E. 1963. Latent infection and premunition. IE} Symposium on immunity to protozoal diseases. Garnham, P. C. C., A. E. Pierce and I. Roitt (Eds.), Blackwell Scientific Publications, Oxford. Sibinovic, K. H., R. McLeod, M. Ristic, S. Sibinovic, and H. W. Cox. 1967. A study of some of the physical, chemical and serologic properties of antigens from sera of horses, dogs, and rats with acute babesiosis. J. Parasitol. 53:919-923. Sibinovic, K. H., R. Milar, M. Ristic, and H. W. Cox. 1969. Ig_vivo and 12 vitro effects of serum antigens of babesial infection and their antibodies on parasitized and normal erythrocytes. Ann. Trop. Med. Parasitol. 63:327-336. Sibinovic, K. H., S. Sibinovic, M. Ristic, and H. W. Cox. 1967. Immunogenic properties of babesial serum antigens. J. Parasitol. 53:1121-1129. Smith, T. and F. L. Kilborne. 1893. Investigations into the nature, causation and prevention of Texas or southern cattle fever. U.S. Dept. Agric. Bur. Ann. Inc. Bull. 1:1-301. Soni, J. L. and H. W. Cox. 1974. Pathogenesis of acute avian malaria. I. Immunologic reactions associated with anemia, splenomegaly and nephritis of acute Plasmodium gellinaceum infections of chickens. Amer. J. Trop. Med. Hyg. 23:577-585. Soni, J. L. and H. W. Cox. 1975a. Pathogenesis of acute avian malaria. II. Anemia mediated by a cold-active autohemagglutinin from the blood of chickens with acute Plasmodium gallinaceum infection. Amer. J. Trop. Med. Hyg. 24:206-213. Soni, J. L. and H. W. Cox. 1975b. Pathogenesis of acute avian malaria. III. Antigen-antibody complexes as a mediator of anemia in acute Plasmodium gallinaceum infections of chickens. Amer. J. Trop. Med. Hyg. 24:423-430. Soni, J. L. and H. W. Cox. 1975c. Pathogenesis of acute avian malaria. IV. Immunologic mediators of nephritis in acute Plasmodium gallinaceum infections of chickens. Amer. J. Trop. Med. Hyg. 24:431-438. 42 Soulsby, E. J. L. 1968. Helminths, arthropods and protozoa of domesticated animals (Mbnnig) 6th Ed. The Williams and Wilkins Company, Baltimore. Sprent, J. F. A. 1963. Parasitism. The Williams and Wilkins Company, Baltimore. Starcovici, C. 1893. Centr. Bakteriol. Parasitenk. Abt. 14:1. Streng, O. 1909a. Studien fiber das verhalten des rinder serums gegenfiber den microbien. Versuch einer neuen serodiag- nostichen method. Zbe. Bakt. (Orig.) 50:47-78. Streng, O. 1909b. Agglutinin oder konglutinin? Zgl. Bakt. (Orig.) 52:523-531. Streng, O. 1930. Immunakonglutinin-antikomplement. Acta. Path. Microbiol. Scand., Suppl. 111 20:411-429. Streng, O. and E. Ryti. 1923. Vergleichende untersuchunger fiber die agglutination und die konglutination der blutkorperchen und bakterium in neutrolsalzlSsungen. Acta. Med. Duodecin. IV. No. 3:1-68. Taliaferro, W. H. 1956. Functions of the spleen in immunity. Am. J. Trap. Med. Hyg. 5:391-410. Tedesco, F., R. Corrocher, and D. L. Brown. 1972. The role of immunoconglutinin in the ig_vivo and ig_vitro destruction of red cells. Clin. Exp. Immunol. 10:685-696. Thompson, P. E. and A. Bayles. 1966. Eradication of Epegythrozoon coccoides with oxophenarsine in normal and drug resistant lines of Plasmodium berghei in mice. J. Parasitol. 52:674-678. Thoongsuwan, S. 1971. Antigenic variants of the haemosporidian parasite, Babesia rodhaini, selected by i§_vitro treatment with immune globulin. M.S. thesis, Michigan State University, East Lansing, Michigan. Thoongsuwan, S. and H. W. Cox. 1973. Antigenic variants of the haemosporidian parasite, Babesia rodhaini, selected by ig_vitro treatment with immune globulin. Ann. Trop. Med. Parasitol. 67:373-385. Thoongsuwan, S. and H. W. Cox. 1976a. Comparative studies of infectious anaemias as in rats. 1. Haemolytic anaemia and glomerulonephritis associated with haemagglutinin in rats infected with a filterable agent. Ann. Trop. Med. Parasitol. (Submitted) 43 Thoongsuwan, S. and H. W. Cox. 1976b. Comparative studies of infectious anaemias in rats. 11. Autoimmune-like anaemia associated with Trypanosoma lewisi infection. Ann. Trop. Med. Parasitol. (Submitted) Udaka, K. 1971. The Arthus reaction. 12; Inflammation, Immunity and Hypersensitivity, H. 2. Movat (Ed.), pp. 390-423. Harper and Rowe, Publishers, New York. Van den Berghe, L., I. H. Vincke, M. Chardome, and M. Van den Bulcke. 1950. Babesia rodhaini N. sp. d'un rongeur du Congo Belge transmissible a la souris blanche. Ann. Soc. Belge. Med. Trap. 30:83-88. von Pirquet, C. F. and B. Schick. 1905. Die Serumkrankheit. Franz Deuticke, Leipzig und Wien. Translated by Dr. B. Schick. 1951. The Williams and Wilkins Co., Baltimore, Md. Wassermann, A., A. Neisser, and C. Bruck. 1906. Dtsch. Med. Wschr. 32:745. Wassermann, A., A. Neisser, C. Bruck, and A. Schuscht. 1906. Z. Hyg. Infekt. Kr. 55:451. Weinman, D. 1944. Infectious anemias due to Bartonella and related red cell parasites. Trans. Am. Phil. Soc. 33:243-287. Wiener, A. S. and L. Klatz. 1951. Studies on the use of enzyme- treated red cells in tests for RA sensitization. J. Immunol. 66:51-66. Wilson, G. S. and A. A. Miles. 1964. Topley and Wilson's Principles of bacteriology and immunity. Vol. 2, pp. 2190-2206. Woodruff, A. W. 1973. Mechanisms involved in anaemia associated with infection and splenomegaly in the tropics. Trans. Roy. Soc. Trop. Med. Hyg. 67:313-328. Woodruff, A. W., E. Topley, R. Knight, and C. G. 8. Downie. 1972. The anaemia of Kala-Azar. Brit. J. Haematol. 22:319-329. Article 1 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS I. HAEMOLYTIC ANAEMIA AND GLOMERULONEPHRITIS ASSOCIATED WITH HAEMAGGLUTININ IN RATS INFECTED WITH A FILTERABLE AGENT Santi Thoongsuwan Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (Submitted to Annals of Tropical Medicine and Parasitology, Liverpool, England) 44 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS I. HAEMOLYTIC ANAEMIA AND GLOMERULONEPHRITIS ASSOCIATED WITH HAEMAGGLUTININ IN RATS INFECTED WITH A FILTERABLE AGENTl’2 Santi Thoongsuwan3 Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 1This communication is from a thesis entitled, "Comparative Studies of Infectious Anemias in Rats," submitted by the author in partial ful- fillment of the requirements for the Ph.D. degree from Michigan State University. His participation in these studies was made possible by a Faculty Fellowship award from the Chulalongkorn University, Bangkok, Thailand. 2This communication is Journal Article No. 7325 from the Michigan Agricultural Experiment Station. 3Present Address: Department of Microbiology Faculty of Pharmacy Chulalongkorn University Bangkok, Thailand 45 46 INTRODUCTION It was observed that a laboratory strain of Trypanosoma lewisi used at this laboratory for teaching purposes, produced signs of haemolytic anaemia, often followed by death, in mature rats. Soulsby (1968) had characterized I, lewisi as a nonpathogenic trypanosome, and the disease signs presently reported differed dramatically from those previously observed (Cox, 1964). It was therefore, suspected that the disease signs observed in this infection might have been in part due to an occult companion agent, as was shown in the case of the laboratory strain of Plasmodium lophurae maintained at this laboratory (Ludford et al., 1969; 1972). A long standing interest in the pathogenesis of infectious haemolytic anaemias prompted us to investigate this suspicion. The study resulted in finding that trypanosome free filtrate of the blood of rats infected with this strain of I, lewisi produced a haemolytic disease with death in mature rats within 5 to 10 days after inocula- tion. For more than a year, rats used to transfer the trypanosome- free agent at weekly intervals have all died of the disease. Methods whereby the agent was isolated, our attempts to visualize and culture it and the disease produced by the agent are described in this communication. 47 MATERIALS AND METHODS Experimental Animals: Male Sprague-Dawley rats and Swiss mice used for the study were purchased from Spartan Research Animals Inc., Haslett, Michigan. Methods of animal care and experimental procedures were consistent with those promulgated by the Institute of Laboratory Animal Resources of the National Research Council. Experimental Infections: The agent was isolated from the blood of rats infected with a Trypanosoma lewisi strain that has been stored in liquid nitrogen in the Department of Microbiology and Public Health (MPH) of Michigan State University. The MPH strain was last used as an experimental infection in Dexamethasane treated rats by Patton and Clark (1968) who did not indicate its origin. A new strain was obtained from American Type Culture (ATC) Collection, 12301 Parklawn Dr., Rockville, Maryland. The MPH and ATC strains of I, legi§i_were maintained by blood passage as described (Cox, 1964) or stored as glycerinated infected blood in liquid nitrogen. For experimental infections, blood of etherized rats infected with I, legi§i_was drawn by cardiac puncture and added 10 parts to 1 of heparinized saline (100 units sodium heparin/ml of 0.85% NaCl solution). After standing for 30 min at 5 C, the blood was centri- fuged at 800 g for 10 min and the plasma removed. The sediment was washed 3 times by suspension in equal volumes of cold saline (0.85% NaCl solution) and centrifugation at 800 g for 10 min. After the 48 final centrifugation the sediment was resuspended in saline and again sedimented by centrifugation. The sedimented cells and supernatant were then placed in a water bath at 37 C for 30 min to allow the trypanosomes to become active. The recovered supernatant contained active trypanosomes and a few blood cells. The number of trypanosomes per cu mm was determined microscopically using Sahli rbc pipettes, Hayems solution and a Neubauer hemocytometer counting chamber, following standard methods using for rbc counts. Infections were standardized to l X 106 trypanosomes per ml. Detection and isolation of the companion agent of the MPH strain of T. lewisi Six mature rats were inoculated intraperitoneally (IP) with 106 trypanosomes of the ATC strain and 6 others with the MPH strain of I, lewisi. Erythrocyte and parasite counts were made daily on all rats until death or recovery from the infections. The six rats recovered from ATC strain infection and 6 normal rats were each inoculated with 106 trypanosomes of the MPH strain. Blood for rbc and trypanosome counts were taken daily. The recovered rats remained trypanosome free but became acutely anaemic on the 7th day. Blood from these rats, diluted 1:100 was inoculated IP into normal rats. The passage rats remained free of trypanosomes and developed anaemia with haemaglobinuria on the 5th or 6th day after infection. The trypanosome-free agent (TFA) has been maintained by passage of 1 m1 of a 1:100 dilution of blood from anaemic to fresh rats at 3-4 day inter- vals. It was also stored as infected glycerinated blood in liquid nitrogen, just as were the I, lewisi strains. 49 When TFA or trypanosomes were needed as experimental infections, rats were inoculated intraperitoneally with the stored blood. The infections were then maintained by blood passage as described (Cox, 1964). Tests to identify TFA Blood and urine of rats infected with TFA were examined by darkfield microscopy for spirochaetes. Wrights and Giemsa stained blood films were examined for haemosporidian parasites and for orga- nisms resembling Haemobartonella. Blood of the anaemic rats was cultured for bacteria, leptospira and mycoplasma using media and procedures routinely employed in the diagnostic microbiology laboratory of the College of Veterinary Medicine of Michigan State University, as recommended (Carter, 1973). Blood from anaemic rats was cultured on three separate occasions using both aerobic and anaerobic methods. A rat embryo cell culture line was initiated as recommended by Roberts and Carter (1972). These cultures were inoculated with blood from anaemic rats and observed daily for cytopathogenic effects. On the 7th day, supernatant of the cultures was transferred to freshly grown cells. Cells grown on cover glasses were fixed with methanol and stained with haematoxylin and eosin to be studied for inclusions and altered cell morphology (Roberts and Carter, 1972). Cells from the 4th culture passage and cells from uninaculated cultures were recovered and disrupted by alternate freezing in methanol-dry ice mixture and thawing in a 37 C water bath. After centrifugation at 800 g for 15 min, 1 ml of the supernatant fluids was inoculated 1? into rats. Red cell counts were made daily for 14 50 days on the 6 rats in each group. Three weeks after inoculation, each rat in each group was inoculated IP with 1 ml of a 1:100 dilution of blood from a rat infected with TFA. Red cell counts were again made daily until all of the rats were dead, or had recovered from anaemia. Tests for infectivity of TFA for mice and for contact transmission to rats Three normal rats and five mice were each inoculated with 1.0 ml of a 1:100 dilution of blood from rats with TFA infection. The rats were housed in the same cage with 3 normal rats. All animals were observed daily for 30 days. Red blood cell counts were made daily on all until death, or for 2 weeks. Weekly rbc counts were made on sur- vivors after the second week. The uninaculated companion rats of the experiment were inoculated with blood from anaemic rats on the 30th day and daily blood counts were continued. Fluorescent antibody tests of blood and spleens from infected rats for TFA Young rats that had recovered from TFA infections were rein- oculated with 1 m1 of blood from anaemic rats. Three weeks later they were exsanguinated by cardiac puncture under ether anesthesia. The serum was recovered and the globulin precipitated twice at 50% of saturation with ammonium sulphate. The globulin was dialized against borate buffered saline, pH 8.4, ionic strength 0.175 until free of sulphate as determined by precipitate from added BaCl solution. The 2 globulin was then adjusted to 25 mg of protein per ml as determined by the methods of Lowry et a1. (1951). It was then conjugated with 51 fluorescein isothiocyanate (FITC) as recommended (Goldman, 1968). The conjugated globulin was filtered through a Sephadex G-25 column and absorbed with activated animal charcoal (Soni and Cox, 1975b). It was then absorbed with 1 ml of washed-packed buffy coat from blood of normal rats per 1 ml of the conjugate at 37 C for 2 hours. The conjugate was stored in small aliquots at -18 C. Blood films and impression slides of the cut surface of spleen from normal rats and rats anaemic from TFA infection were fixed for 3 min in absolute methanol. The slides were then incubated with the FITC conjugated globulin diluted 1:4 with 0.01 M phosphate buffered 0.85% NaCl solution, pH 7.5 (PBS) at 37 C for 30 min. The slides were rinsed in 3 changes of PBS and a #1 cover glass was mounted with 90% glycerine in PBS. The slides were examined for fluorescent activity with a Zeiss Fluroscope. Photomicrographs were made with Kodak Tri-X film at an exposure time of 90 sec. Filtration experiments Whole blood from rats infected for 4 days with TFA was added to heparinized saline as described and subjected to freeze-thaw treatment using a dry ice and methanol mixture and thawing in a 37 C water bath. After two treatments the fluids were clarified by centri- fugation at 800 g for 20 min. The supernatant was diluted 1:10 with saline and divided into equal portions. One portion was passed through a 0.20 micron Nalgaline filter unit (Nalge Sybron Corporation, Rochester, NY) and the other through a 0.45 micron membrane (Becton, Dickinson 8 Co., Oxnard, CA). Samples of each filtrate were inoculated to Myocoplasma medium obtained from Difco Laboratories, Detroit, 52 Michigan and incubated for 7 days at 37 C in a high moisture and C02 atmosphere. The remaining filtrates were each diluted 1:10 and 1 ml of each was injected into each of 8 rats for the 0.45 micron filtrate and 8 for the 0.2 micron filtrate. RBC counts were made daily on each rat . Tests for splenomegely and erythrephagecytosis in rats infected with the MPH strains of T. lewisi and with TFA In all seven normal mature rats, six mature rats used for passage of the MPH strain and eight used for TFA passage were autopsied and the spleen was measured in ml by volume displacement of saline in a graduate cylinder. Each spleen was cut transversely and impression smears were made of the cut surface. After the smears had air dried, they were fixed for 3 min in absolute methanol and stained for 30 min in Giemsa stain diluted 1:10 with 0.01 M phosphate buffer, pH 6.8. These preparations were examined for phagocytosis of erythrocytes and trypanosomes by splenic macrophages. Smear preparations were made from bone marrow obtained by splitting the head of the femur. These were fixed and stained as described and also examined for erythrophagocytic activity. Histologic study of kidneys from rats made anaemic by TFA infection Kidneys were taken from 6 anaemic rats sacrificed for spleen studies. They were cut into small pieces 2-3 mm thick and fixed in 10% formalin. The tissues were cleared, dehydrated and embedded. Sections were cut at 4 microns and stained with hematoxylin and eosin (Luna, 1968). Kidney histopathology of the glomeruli and convoluted 53 tubules was evaluated following criteria used for babesial and malarial nephritis (Iturri and Cox, 1969; Soni and Cox, 1974). Photomicrographs of kidney, spleen and bone marrow were made with a Zeiss Fluroscope by tungsten filament illumination with a blud filter using Kodak High Speed Daylight Ektachrome film at an exposure of 0.25 sec. Red cell and leucocyte counts, parasitaemia, and cold-active haemeg- glutinin (CAH) in rats inoculated with normal rat erythrqeytes, and rats infected with TFA Two groups of 14 young rats were used. The first group was inoculated with 109 normal rat blood cells per rat. Each rat of the second group was inoculated with 1 m1 of a 1:100 dilution of blood from a rat infected with TFA. Six rats from each group furnished blood samples daily for wbc and rbc counts. The other eight in each group were divided into groups of four rats. These groups were bled by cardiac puncture on alternate days throughout the experiment to furnish daily serum samples to be tested for CAH as described (Thoongsuwan and Cox, 1973). Rbc and wbc counts were made from blood drawn from the tip of the tail after it was snipped with scissors. Samples were taken with standard Sahli hemocytometer pipettes using Hayems fluid for rbc counts, and 2% glacial acetic acid as diluent for wbc counts. The counts were made microsc0pically using a Neubauer hemocytometer count- ing chamber. Blood films for differential leucocyte counts were stained with Wrights Stain. 54 Comparison of anaemia and mortality from TFA infections in old (300 gm) and young (100 gm) rats A group of eight young rats and eight mature rats were each given the standard inoculum of TFA infected rat blood. Red cell counts were made daily on these rats until death or recovery. EXPERIMENTAL RESULTS Comparison of parasitaemia and anaemia in rats infected with the MPH and ATC strains of T. lewisi The data from groups of six rats are shown, Table l. Reduc- tions in red cell counts of rats with MPH strain infections were noticed on day 6 or 7. On day 8 the average cell counts of these rats was less than 3 X 106 and most of the rats exhibited haemaglobinuria with urine the color of Port wine. Four of these rats were dead on day 9. The remaining 2 rats had recovered from both anaemia and parasitaemia by day 20. Reduction in cell counts of rats infected with the ATC strain were evident on day 7, and counts were lowest on day 9. These rats did not exhibit haemaglobinuria and all had recovered by day 24. Trypanosomal parasitaemia was lower in rats infected with MPH strain than it was in those given the ATC strain. Parasitaemia and anaemia in normal rats and rats recovered from infection with the ATC strain after infection with the MPH strain of T. lewisi The data from the 6 experimental and 6 control rats are pre- sented, Table 2. Rats that had recovered from the ATC strain of I, 55 lewisi did not show trypanosomal parasitaemia throughout the experi- ment. However, all of the rats began to show anaemia on the 4th day which was severest on day 7. None of these rats died and all appeared to have recovered on day 14. The control rats had detectable trypano- somes in their blood on day 1. Parasitaemia was maximal on day 5 when anaemia was first evident. Blood cell counts fell rapidly and all died with signs of haemaglobinuria before the 8th day. Attempts to detect and identify TFA Study of blood from rats infected with TFA using darkfield illumination and study of Wright's or Giemsa stained blood films did not reveal the presence of spirochaetes, haemosporidian, or Haemo- bartonella. Repeated cultures of blood from anaemic rats for bacteria, leptospira and mycoplasma were negative. Rat embryo cell cultures inoculated with blood and saline washings from minced spleen from anaemic rats did not reveal evidence of cytopathogenic effect or virus-like inclusions in cells through 5 subinoculations. Injection of supernatant from frozen-thawed cells inoculated with the 2nd and 4th passage culture supernatant did not produce anaemia in susceptible rats. Three weeks later when these rats were inoculated with TFA infected blood, they were as susceptible as rats inoculated with the supernatant from control cell cultures. Results of tests for infectivity of filtrates of lysed blood from anaemic rats that had passed 0.45 and 0.2 micron millipore filters are shown, Table 3. Rats inoculated with material from the 0.45 micron filter all died of anaemia 7 days after injection, indicating that this filtrate was as infective as unfiltered blood. Signs of anaemia 56 in rats injected with the filtrate from the 0.2 micron filter did not develop until the 11th day, but they too all died of haemolytic anaemia by the 13th day. Thus, it was indicated that TFA particle was less than 200 nm in diameter. When tested with FITC conjugated globulin from rats hyper- immunized after recovery from TFA infection, blood leucocytes gave bright fluorescence. In some of the cells small dense areas gave intensive reactions. Cells of the spleen sinusoids reacted with "flare-like" fluorescence. No fluorescent activity was seen in blood and spleen preparations from normal rats, Figure 1. Mice inoculated with diluted blood from rats made anaemic by TFA infection did not develop any signs of anaemia while control rats given the same material died with haemolytic anaemia within 8 days. Uninoculated rats housed in the cage with the infected rats did not develop signs of anaemia and appeared normal at the end of the observation period. These rats developed haemolytic anaemia and died within 8 days after they were finally inoculated with blood from anaemic rats. Red blood cell counts, white blood cell counts, and titers of cold- active haemagglutinin in control rats injected with 109 normal rat cells, and rats inoculated with TFA Red cell counts on the control rats increased over the 30 day period as the rats matured. The counts from rats infected with TFA fell sharply from the 4th through the 6th day. There were no deaths and all had recovered from anaemia by day 30. Rats infected with TFA 57 had high titers of CAH from the 2nd through the 5th day. CAH was not detected in plasma samples from the control rats. Table 4. Leucocyte counts were elevated on day 4 in the infected rats and remained so until day 14. Most of the leucocytosis appeared to be due to increases in the number of mononuclear cells, particularly monocytes. Leucocytes resembling those seen in myelogenous or lymphatic leukemia were not seen. Comparison of anaemia and mortality inyoungand old rats infected with TFA The data on rbc counts and mortality in this experiment are summarized, Table 5. Reductions in rbc counts occurred a day earlier in young rats than in mature, but did not drop as precipitously or as low. None of the young animals died, and all appeared to have recovered at the end of the experiment. The rapid reduction in rbc counts of the older rats was accompanied by haemaglobinuria with urine the color of Port wine. Five of these rats died on the 5th day and the remainder on the 6th. Splenomegaly and erythrophegocytosis in rats infected with the MPH strain of T. lewisi and with TFA The folumes of spleens from rats with the infections did not differ, however they were approximately 3 X those of normal rats, Table 6. Phagocytosis of trypanosomes was not seen, however, erythro- phagocytosis in the sinuses of the spleens from rats infected with MPH I, lewisi and with TFA was extensive, Figure 2. Phagocytosis of 58 erythrocytes by bone marrow macrophagocytosis in rats infected with MPH I, lewisi and TFA was as marked as in the spleen, Figure 3. Histolpgy_of kidneys of rats infected with TFA In kidneys from rats with anaemia from TFA infection changes in glomeruli varied from swelling of the glomerular tuft with moderate hypercellularity to nearly complete necrosis. The tuft was often swollen to the extent that it completely occupied Bowmans capsule, which had a thickened wall. The epithelium of the convoluted tubules was often swollen to the extent that the lumen of the tubules appeared to be no longer patent. In others there was necrosis of the epi- thelium. The basement membrane of tubules was usually swollen and interstitial edema was evident. Some tubules contained clear deposits resembling hyaline casts, Figure 4. DISCUSSION These experiments indicated that the haemolytic anaemia and mortality in rats infected with the MPH strain of I, lewisi was due to an agent other than the trypanosome. The absence of bacteria, mycoplasma, spirochetes, Haemosporidia, or Haemobartonella-like orga- nisms indicated that these agents were not involved. Infectivity of freeze-thaw lysed blood that had passed a 0.2 millimicron filter indicated that the particle size of the TFA was less than 200 nm. Tests of blood films from anaemic rats with FITC conjugated globulin from hyperimmunized rats indicated that leucocytes might be infected. In some of these cells small areas of approximately 0.5 u 59 or less in size showed particularly intensive fluorescence. While it was considered possible that these small bodies might have been viral inclusions, we are not prepared to make such a conclusion at this time. Attempts to grow the agent in rat embryo cell cultures (and later in embryonated chicken eggs) were disappointing, as were also attempts to visualize the agent by electron-microscopic study of ultra thin sections of pelletized buffy coat from infected rat blood. Until the agent has been visualized or cultured, we would only suggest that it might be a virus. For convenience sake we have referred to the disease as rat infectious anaemia (RIA). The origin of RIA agent is unknown. The MPH strain of I, lewisi was last used as an experimental infection in Dexamethasane treated rats (Patton and Clark, 1968). In examining their data it appeared that the parasitaemia in their untreated controls did not differ from previously observed data (Cox, 1964). We have given rats infected with the ATC strain of'I, lewisi Dexamethasane treatments used by Patton and Clark (1968), but were unable to obtain similar results. More importantly, we were unable to detect signs of RIA agent after treatment of rats infected with the ATC strain. It therefore, appeared that the ATC strain, which had been donated to the American Type Culture Collection by Dr. Robert G. Yaeger of the Tulane Univer- sity School of Medicine, was not contaminated with RIA agent (unpub- lished). A review of the literature has revealed only one account of anaemia in rats associated with a filterable agent. Infection of neonatal rats with Friends lymphatic leukemia virus caused a runting 60 syndrome associated with a high incidence of haemolytic anaemia which was accompanied by antibody to erythrocytes (Kuzumoki et al., 1974). While we did not infect neonatal rats, we have not seen signs of runting, lymphoma or chronic anaemia in young rats after infection with RIA agent. In mature rats RIA was typically an acute haemolytic anaemia with haemaglobinuria and death within 5-8 days following inoculation. Anaemia was accompanied by a markedly enlarged spleen, extensive phagocytosis of erythrocytes by splenic and bone marrow macrophagocytes, and by high titers of CAH. These disease signs were accompanied by an acute glomerulonephritis involving both glomerular and tubuler elements of the kidney. Necrosis of glomeruli and tubuler epithelium was observed with considerable frequency. In young rats anaemia was less severe, haemaglobinuria was less evident, and they usually recovered within 3-4 weeks. Since recovered rats did not develop haemolytic anaemia following reinoculation, it can be assumed that they had an acquired resistance. These observations add RIA virus, to the list of agents that produce anaemia with splenomegaly accompanied by autoantibody to erythrocytes (Cox and Iturri, 1976; Cox et al., 1966; Oki and Miura, 1970; Schroeder et al., 1966; Schroeder and Ristic, 1965). Since the agent appeared not to be infective for mice, it was not transmitted from infected to normal rats housed in the same cage, and the authors have suffered no ill effects after handling the agent for 2 years. It may serve as a safe and useful model for the study of viral anaemia. 61 SUMMARY A replicating filterable agent isolated from the blood of rats infected with a strain of I, lewisi kept at this Department caused acute haemolytic anaemia, splenomegaly, glomerulonephritis and death within 5-8 days in mature rats. The disease was less severe in wean- ling rats which usually recovered within 3-4 weeks. The anaemia was accompanied by phagocytosis of erythrocytes by monocytes of the spleen and bone marrow and by high titres of cold-active haemagglutinin. In fluorescent antibody tests leucocytes from the blood of anaemic rats reacted strongly with globulin from hyperimmunized recovered rats, revealing the presence of small intranuclear inclusions in some cells. Filtrates of blood from anaemic rats passing a 0.20 micron filter caused anaemia and death of inoculated rats 3-4 days later than did blood filtrates passing a 0.45 micron millipore filter. Thus the particle size of the agent was smaller than 200 millimicrons. Attempts to grow the agent on rat embryo fibroblast cultures were unsuccessful; however, the failure to detect other infectious organisms allows the suggestion that the causal agent for this disease which we have called rat infectious anaemia, might be a virus. REFERENCES 1. Carter, G. R. 1973. Diagnostic procedures in veterinary micro- biology. 2nd ed., Charles C. Thomas, Publisher, Springfield, Illinois. 62 Cox, H. W. 1964. Immune response of rats and mice to trypanosome infection. Journal of Parasitology_ 50:15-22. Cox, H. W., and G. C. Iturri. 1976. Autoimmune factors associated with anaemia in acute Haemobartonella and Eperythrozoon infections of rodents. Annals of Tropical Medicine and Parasitology_ 70:73-70. Cox, H. W., W. F. Schroeder, and M. Ristic. 1966. Hemagglutination and erythrophagocytosis associated with the anemia of Plasmodium bepghei infections of rats. Journal of Protozoolegy 13:327-332. Goldman, M. 1968. Fluorescent antibody methods. Academic Press, New York, 103-104. Iturri, G. M., and H. W. Cox. 1969. Glomerulonephritis associ- ated with acute haemosporidian infection. Military Medicine (Special Issue) 134:1119-1128. Kuzumaki, N., T. Kodama, N. Takeichi, and H. Hobayashi. 1974. Friend lymphatic leukemia virus-induced autoimmune hemolytic anemia with glomerulonephritis in the rat. International Journal of Cancer 14:483-492. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry 193:265-275. Ludford, C. G., R. M. Corwin, H. W. Cox, and T. A. Sheldon. 1969. Resistance of ducks to a Plasmodium sp. induced by a filterable agent. Military Medicine (Special Issue) 134:1276-1283. 63 10. Ludford, C. G., H. W. Purchase, and H. W. Cox. 1972. Duck infectious anemia virus associated with Plasmodium lophurae. Experimental Parasitology_ 31:20-38. 11. Luna, L. G. 1968. Manual of histological staining methods of the Armed Forces Institute of Pathology. 3rd Ed., McGraw Hill Book Co., New York. 12. Oki, Y., and K. Miura. 1970. Characteristics and roles of red cell antibodies in equine infectious anemia. Japanese Journal of Veterinary Research 32:217-227. 13. Patton, C. L., and D. T. Clark. 1968. Trypanosoma lewisi infections in normal rats and rats treated with dexamethasone. Journal of Protozoolegy. 15:31-35. 14. Roberts, A. W., and G. R. Carter. 1972. Simplified and economical cell culture techniques for laboratories beginning clinical veterinary virology. Journal article 5677, Michigan State Agricultural Experiment Station, Michigan State University, East Lansing, MI 48824. 15. Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Anaemia, parasitaemia, erythrophagocytosis, and haemagglutinins in Babesia rodhaini infections. Annals of Tropical Medicine and Parasitology_ 60:31-38. 16. Schroeder, W. F., and M. Ristic. 1965. Anaplasmosis. XVII. The relationship of autoimmune processes to anemia. American Journal of Veterinarpresearch 26:239-245. 64 17. Soni, J. L., and H. W. Cox, 1974. Pathogenesis of acute avian malaria. I. Immunologic reactions associated with anemia, splenomegaly, and nephritis of acute Plasmodium gallinaceum infections of chickens. American Journal of Tropical Medicine and Hygiene 23:577-585. 18. Soni, J. L., and H. W. Cox. 1975. Pathogenesis of acute avian malaria. II. Anemia mediated by a cold-active autohemagglutinin from the blood of chickens with acute Plasmodium gallinaceum infections. American Journal of Tropical Medicine and Hygiene 24:206-213. 19. Soni, J. L., and H. W. Cox. 1975. Pathogenesis of acute avian malaria. III. Antigen-antibody complexes as a mediator of anemia in acute Plasmodium gellinaceum infections of chickens. American Journal of Trgpical Medicine and Hygiene 24:423-430. 20. Soni, J. L., and H. W. Cox. 1975. Pathogenesis of acute avian malaria. IV. Immunologic mediators of nephritis in acute Plasmodium gellinaceum infections of chickens. American Journal of Tropical Medicine and Hygiene 24:431-438. 21. Thoongsuwan, S., and H. W. Cox. 1973. Antigenic variants of the haemosporidian parasite Babesia rodhaini selected by ip_vitro treatment with immune globulin. Annals of Tropical Medicine and Parasitology 67:373-385. 65 Table 1.--Mean number of red blood cellg per mm35(RBC X 106), mean number of trypanosomes per mm (T X 10 ) and standard error of the mean (S.E.) in blood of mature rats infected with the Microbiology and Public Health (MPH) and American Type Culture (ATC) strains of Trypanosoma lewisi. Days RBC X 106 a 5.5. . T x 10 i S.E. . MPH strain ATC strain MPH strain ATC strain 0 8.63 i 0.29 8.33 i 0.21 -- - l 8.96 i 0.23 8.92 i 0.11 -- - 2 9.13 i 0.34 8.73 i 0.18 t 3 8.72 i 0.26 8.53 i 0.15 0.04 i 0.01 .03 i 0.01 4 8.96 i 0.26 9.14 i 0.10 0.18 i 0.03 .41 i 0.04** 5 8.36 i 0.08 8.41 i 0.17 0.72 i 0.19 .99 t 0.44 6 7.91 i 0.11 8.26 i 0.25 1.36 i 0.42 .02 i 0.91** 7 6.92 i 0.38 7.55 i 0.33 1.58 i 0.49 .62 i 1.21** 8 2.98 i 0.26 7.03 i 0.41** 1.80 i 0.59 .24 i 1.12 9 2.20 i 0.05* 6.89 i 0.38** 0.11 i 0.01* .77 i 1.25 10 3.20 t 0.10 7.03 i 0.46** 0.14 i 0.04 .44 i 0.88 11 3.67 i 0.16 7.15 i 0.48** 0.16 i 0.10 .25 i 0.73 12 5.52 i 0.16 7.43 i 0.39** 0.08 i 0.02 .31 i 0.79 13 6.44 i 0.18 8.17 i 0.33** 0.06 i 0.02 .05 i 0.83 15 6.93 i 0.31 7.98 i 0.39 0.02 t 0.01 .53 i 0.64 17 7.26 i 0.24 7.88 i 0.31 0.08 i 0.02 .04 i 0.73 20 8.67 i 0.14 8.01 i 0.41 -- .36 i 0.60 24 8.99 i 0.07 9.28 i 0.13 -- - TRare trypanosomes found. *Four of 6 rats dead on 9th day. There was no mortality among the 6 rats of the group infected with the ATC strain. **Significant at P < 0.05 or less (Students t). 66 'Table 2.--Mean and standard6error of the mean (S.E.) of red blood cells per mm3 (RBC X 10 ) and the mean and S.E. of the number of trypanosomes per mm (T X 105) in normal mature control rats, and mature rats recovered from infection with the American Type Culture (ATC) strain of Trypanosoma lewisi, after infection with the Microbiology and Public Health (MPH) strain of I, lewisi. Normal Rats p Recovered Rats Days RBC x 106’: S.E. T x 10“‘: S.E. RBC x 10U i S.E. T x 105 0 9.96 i 0.29 -- 9.28 i 0.35 --- l 9.58 i 0.31 + 9.53 i 0.28 --- 2 9.52 i 0.23 I 9.59 i 0.30 --- 3 10.19 i 0.26 0.057 t 0.01 9.73 x 0.30 _-- 4 9.54 t 0.33 0.567 i 0.19 8.82 1 0.20 --- 5 7.11 t 0.31 1.103 t 0.39 7.73 i 0.31 --- 6 4.58*: 0.48 0.934*: 0.56 4.57 i 0.83 --- 7 2.54 i 0.22 0.907 t 0.65 2.98 i 0.28 --- 8 4.19 a 0.32 --- 10 5.54 z 0.26 --- 12 6.63 i 0.16 --- 14 8.57 i 0.17 --_ +Rare trypanosomes found. *First mortality on day 6. Remainder of the 6 rats were dead on day 8. No mortality among the 6 recovered rats. 67 Table 3.--Mean number of red blood cells per mm3 (RBC x 106) and standard error of the mean (S.E.) of 8 mature rats after inoculation with the supernatant of freeze-thaw treated whole blood of rats infected with TFA that had been passed through a 0.20 micron (200 nm) Millipore membrane, and 8 mature rats inoculated with the supernatant that passed through a 0.45 micron (450 nm) membrane. Days . RBC x 106 i S.E. . 200 nm filtrate 450 nm filtrate 0 8.32 t .09 8.43 i 0.16 l 8.44 i .10 8.56 i 0.14 2 8.49 i .09 8.65 i 0.12 3 8.60 i .12 8.64 i 0.14 4 8.48 i .12 8.40 i 0.11 5 8.68 i .13 8.57 i 0.16 6 8.43 i .12 6.88 i 0.43 7 8.64 i .14 2.77 i 0.12* 8 8.73 i .16 9 8.63 i .19 10 8.24 t .16 11 5.39 i .84 12 3.04 i .29** d 8 *Four of 8 rats in this group died on day 7, the remainder on ay . **Five of 8 rats in this group died on day 12, the remainder on day 13. 'Table 4.--Mean and standard per mm 6 5 68 error of the mean (S.E.) of red blood (RBC X 10 ), mean number of trypanosomes per mm cells (T X 10 ) and the mean titres of cold-active haemagglutinin (CAH) in blood of young rats injected with normal rat blood cell suspension (Group I), and rats infected with TFA (Group II). Group I Grouppll Days RBC x 106: S.E. T x 105 CAH RBC x 106 : S.E. T x 105 CAH 0 5.68 t 0.16 --- --— 5.93 i .14 --- --- 1 6.26 i 0.27 --- --— 6.32 i .21 --- --- 2 6.53 i 0.25 --- --- 6.73 1 .14 --- 256* 3 6.79 i 0.23 --— --- 6.39 1 .19 --- 256 4 6.83 i 0.25 --- --- 4.23 t .43 --- 256 5 7.05 i 0.24 --— --- 2.89 i .08 --- 256 6 7.35 2 0.06 --- --- 3.31 i .23 --- --- 7 7.18 i 0.22 --- --- 4.42 i .18 --- --- 9 7.21 i 0.24 --— -—- 5.29 1 .18 --- --- 11 7.26 i 0.36 --- --- 5.74 i .17 --- --- 14 7.44 i 0.14 --- --- 6.55 i .13 --- -_- 17 7.64 a 0.12 --- --- 7.59 i .16 --- --- 20 7.86 i 0.07 --- --- 7.61 a .24 ——- --- 24 7.75 i 0.09 --- --- 7.68 i .13 --- --- 30 7.95 i 0.11 --- --- 7.93 z .12 ——- --- *CAH titres were all 256 or higher. 69 'fable S.--Mean red blood cell counts (RBC X 106), i standard error of the mean (S.E.) and mortality among 8 young rats and 8 mature rats after infection with TFA. Days RBC x loéyguggegats No. Deaa' RBC x logaiug?egats No. Dead 0 6.12 1 0.18 0 9.54 1 0.11 0 1 6.29 1 0.09 o 9.23 1 0.32 0 2 6.44 1 0.11 0 9.86 1 0.26 0 3 6.81 1 0.14 0 9.47 1 0.22 o 4 4.54 1 0.33 0 9.34 1 0.37 0 5 2.77 1 0.42 0 7.27 1 0.46 0 6 2.92 1 0.12 0 2.61 1 0.34 5 7 3.81 1 0.23 0 8 9 4.63 1 0.16 0 11 5.76 1 0.24 0 14 6.48 1 0.11 0 17 7.13 1 0.22 0 20 7.62 1 0.09 0 24 7.96 1 0.12 0 30 8.14 1 0.08 0 70 Table 6.--Average spleen volume in ml of normal rats, rats infected with the Microbiology and Public Health (MPH) strain of Trypanosoma lewisi, and rats infected with TFA found as a companion of the MPH strain of I, lewisi. Number Average Vol. in ml Range Normal rats 7 1.01 0.9 - 1.2 Infected with MPH strain 6 3.07 2.8 - 3.2 Inf°°t°d "1th 8 3.04 2.4 - 3.8 TFA 71 Fig. l.--Photomicrographs of blood films and spleen impression slides from normal rats and rats with TFA infection after incubation with FITC conjugated globulin from rats hyperimmunized by TFA infections. A. Blood film from a normal rat (125X). B. Blood film from a rat with acute TFA infection (125X). Note the fluorescence of the cytoplasm of the polymorphonuclear (PMN) leucocytes, and the small intranuclear areas of fluor- escence seen in some. C. PMN leucocytes showing fluorescence of intranuclear inclusions (lOOOX). D. Fluorescence of leucocytes in the spleen from a rat with acute TFA infection (125X). 72 Figure 1 73 Fig. 2.--Photomicrographs of Giemsa stained spleen impression slides (lOOOX). A. Slide from normal rat spleen. B. Slide from the spleen of a rat infected with the ATC strain of I, lewisi. C. Slide from the spleen of a rat infected with the MPH strain of I, lewisi. D. Slide from the spleen of a rat infected with TFA. Phagocytosis of trypanosomes was not observed. MacroPhagocytes engorged with erythrocytes were abundant in the preparations from rats infected with MPH I, lewisi and TFA. 75 Fig. 3.--Photomicrographs of Giemsa stained bone marrow films (lOOOX). A. Preparation from a normal rat. B. Preparation from a rat infected with the ATC strain of I, lewisi. C and D. Preparations from a rat with TFA infection. Phagocytized trypanosomes were not seen, and macrophagocytes engorged with erythrocytes were abundant in preparations from TFA infected rats. 76 77 Fig. 4.--Photomicrographs of rat kidney sections cut at 4 u and H and E stained (700X). A. View of glomerulus and adjacent con- voluted tubules from a normal rat kidney. B-F. Views of sections from kidney of a rat with acute TFA infection. B. Note swelling and hypercellularity of the glomerular tuft, and that the lumen of the adjacent tubules are obliterated due to swelling of the epithelium. C. Note fat deposits adjacent to the tuft and the thickening of Bowman's membrane. D. Necrosis with the loss of structure of the capillary loop was a frequent finding. E. Desquamation of the tubular epithelium and masses resembling hyaline casts were found. F. Note interstitial edema and thickened basement membrane, found about the distal convoluted tubules. 78 Figure 4 Article 2 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS II. AUTOIMMUNE-LIKE ANAEMIA ASSOCIATED WITH TRYPANOSOMA LEWISI INFECTION Santi Thoongsuwan Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (Submitted to Annals of Tropical Medicine and Parasitology) (Liverpool, England) 79 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS II. AUTOIMMUNE-LIKE ANAEMIA ASSOCIATED WITH TRYPANOSOMA LEWISI INFECTIONI’Z Santi Thoongsuwan3 Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 1This communication is from a thesis entitled "Comparative Studies of Infectious Anemias in Rats" submitted by the author in partial ful- fillment of the requirements for the Ph.D. degree from Michigan State University. His participation in these studies was made possible by a Faculty Fellowship award from the Chulalongkorn University, Bangkok, Thailand. 2This communication is Journal Article No. 7593 from the Michigan Agricultural Experiment Station. 3Present Address: Department of Microbiology Faculty of Pharmacy Chulalongkorn University Bangkok, Thailand 80 81 INTRODUCTION A disease syndrome of anaemia with splenomegaly and vascular- renal disease accompanied by autoimmune-like blood factors may be associated with the infections of unrelated agents (Cox and Iturri, 1976; Cox et al., 1966; Iturri and Cox, 1969; Oki and Miura, 1970; Schroeder et al., 1966). The association of auto-antibody and the absence of a specific etiologic agent led to a suggestion that this infection-associated syndrome should be called infectious anaemia (Cox and Iturri, 1976). The authors reported that a replicating filterable agent found as a companion of a laboratory strain of Trypanosoma lewisi in the blood of rats caused acute haemolytic anaemia, splenomegaly with erythrophagocytosis and acute glomerulonephritis which was accompanied by high.titres of cold-active haemagglutinin (HA). Since other infectious agents were not detected and the companion agent passes a 0.2 micron filter, it was suggested that it might be virus. The disease resulting from infection was called rat infectious anaemia (RIA) (Thoongsuwan and Cox, 1977). In the course of liberating RIA agent from its trypanosome companion it was noticed that the new (ATC) strain of I, lewisi obtained from the American Type Culture Collection produced a moder- ately severe and persistent anaemia. It differed from the lethal haemolytic anaemia associated with infections of the contaminated strain and from RIA in that it was not haemolytic and none of the rats died. Since I, Igwisi had been characterized by Soulsby (1968) as "nonpathogenic" for mature rats, it was felt that infection of 82 rats with this parasite deserved further investigation. It was found that there was a persistent anaemia with splenomegaly and signs of glomerulonephritis, accompanied by the presence of HA, associated with the late phase of infection with the ATC strain of I, lewisi. The details of these experiments are presented and discussed. MATERIALS AND METHODS Animals and infections The strain and source of rats, the methods of animal care, the source and maintenance of the ATC strain of I, lewisi, and the methods for standardizing I, lewisi infections were described (Thoongsuwan and Cox, 1977). Trypanosomal parasitaemia and anaemia in rats were determined by microscopic methods using Sahli haemocytometer pipettes with Hayem's diluting fluid and a Neubauer haemocytometer counting chamber as described (Thoongsuwan and Cox, 1977). Tests for cold-active haemagglutinin (HA), Rats were bled by cardiac puncture under ether anaesthesia. Three m1 of blood was withdrawn and added to 0.3 ml of heparinized saline (100 units sodium heparin per ml of 0.85% NaCl). The plasma was harvested and stored at -18 C until the samples could be tested for HA with trypsinized rat erythrocytes as described (Thoongsuwan and Cox, 1973). 83 Studies for splenomegaly,erythrophagocytosis andIglomerulonephritis Experimental and control rats sacrificed for autopsy were exsanguinated by cardiac puncture under ether anaesthesia and the organs removed. Spleen volume was determined by fluid volume dis- placement as described (Soni and Cox, 1974). The organ was then cut transversely and impression slides were prepared, stained by the Giemsa method and examined for erythrophagocytosis (Thoongsuwan and Cox, 1977). The kidneys were cut into small pieces which were fixed in 10% formaldehyde. These were cleared, mounted, cut and stained with haematoxylin-eosin following standard procedures (Luna, 1968). Pathologic changes were evaluated by criteria of a swollen glomerular tuft, swelling of tubuler epithelium, edema of Bowman's membrane and tubular basement membrane. To obtain a numerical value for the severity of kidney damage (SKD), 100 randomly selected nephrons (glomerulus and adjacent convoluted tubules) were evaluated from 0 to 4 plus. The sum of the evaluations represented the SKD score for the animal (Iturri and Cox, 1969). Hypercellularity of glomeruli was used as a second parameter. Counts were made of the number of nuclei in the glomerular tuft (NGT) of 100 randomly selected glomeruli (Kibukamusoke and Hutt, 1967). Comparison of the means of the counts from infected and control rats were subjected to statistical analysis (students to test). Microphotography Photomicrographs of kidney sections and spleen impression slides were made with a Zeiss Fluroscope with tungsten filament 84 illumination and a blue filter using Kodak High Speed Daylight Ectachrome film and an exposure time of 0.25 sec. Erythrocyte and parasite counts and titers of cold-active haemagglutinin (HA) during the course of T. lewisi infection in rats Two groups of 14 rats were used. Rats of the control group were each inoculated intraperitoneally (IP) with 1 x 109 normal rat blood cells. Rats of the 2nd group were each inoculated with 1 x 106 trypanosomes separated from infected rat blood as described (Thoongsuwan and Cox, 1976). Six rats from each group were used to fUrnish blood for rbc and trypanosome counts. The remaining 8 of each group were divided into groups of 4 rats. One group of 4 infected and 4 control rats were bled on days 0, 5, 9, l4 and 20, and the other groups of 4 were bled on days 3, 7, ll, 17 and 24 so that plasma samples from infected and control rats were taken at 2-day intervals without excessive stress on the animals. Plasma samples recovered from the blood were stored at -18 C until they could be tested for HA with trypsinized rat erythrocytes. EXPERIMENTAL RESUETS The means of rbc counts and trypanosome counts and the mean titres of the HA are presented graphically (Figure 1). As these rats were young, the rbc counts of the controls which were injected with normal rat erythrocytes, gradually increased from approximately 6 x 6 106/cu mm to near 8 x 10 at the end of the experiment. HA was detected first on day 5 near the time when a significant drop in the 85 rbc counts was evident. Both HA and anaemia persisted throughout the period of chronic parasitaemia. Recovery from anaemia was associated with recovery from parasitaemia and the disappearance of HA from the blood of the rats. HA was not detected in plasma from any of the control rats of the experiment. The spleens removed from rats with I, lewisi anaemia were engorged, firm and approximately three times the volume of spleens removed from normal rats and the kidneys were also enlarged. Urine collected from the rats did not show gross evidence of haemaglobinuria. In the impression slides, areas of red and white pulp were packed with erythrocytes. Trypanosomes were present but not in numbers greater than those seen in blood. Phagocytized erythrocytes but not trypanosomes were found occasionally in splenic macrophagocytes. In sections of kidneys from rats with I, lewisi anaemia the glomerular tuft was swollen to the extent that Bowman's capsule was almost completely occupied. The number of mesengial nuclei of the tuft was increased from a normal of 40-50 to 70-80. Fibrinous adherence of the tuft to Bowman's membrane was evident and the membrane itself was thickened. The epithelium of convoluted tubules adjacent to glomeruli was swollen to the extent that the lumen of the tubules appeared to be closed (Figure 2). Abnormal numbers of hyaline casts were observed in the distal convoluted tubules. The means of the SKD scores and of NGT counts and mean spleen volumes for control and experimental rats are summarized in Table 1. 86 DISCUSSION These experiments indicate that I, lewisi is pathogenic for laboratory rats. During the post-acute phase of the infections rats suffer moderate to severe anaemia with splenomegaly and mild signs of glomerulonephritis accompanied by the presence of cold-active haemag- glutinin detected with trypsinized rat erythrocytes. The onset of anaemia was sudden without visible signs of haemolysis. It then became chronic, persisting with gradual recovery for as long as parasitaemia and the haemagglutinin were detected. All rats appeared to have recovered from the disease by the 30th day after infection. The signs of nephritis were primarily swelling of endothelial cells of the capillary loop, hypercellularity of the glomerular tuft, swelling of tubular epithelium, edema of basement membrane and extravasation of proteinaceous matter as indicated by the hyaline casts. Except that they were milder, these signs resembled those associated with anaemia in Babesia rodhaini infections of rats, RIA and Plasmodium gallinaceum infections of chickens, all of which were associated with the presence of cold—active haemagglutinin (Iturri and Cox, 1969; Soni and Cox, 1974; Thoongsuwan and Cox, 1977). The relevance of cold-active haemagglutinin to the anaemia or nephritis associated with infections of rodents has not been indicated. However, its association with anaemia from infections with RIA agent, babesiosis, malaria, haemobartonellosis and eperythrozoonosis in rats or mice has been established (Cox and Iturri, 1976; Cox et al., 1966; Iturri and Cox, 1969; Oki and Miura, 1970; Schroeder et al., 1966; Thoongsuwan and Cox, 1977). Soni and Cox (1975) indicated that the 87 agglutinin associated with anaemia of chicken malaria was auto- antibody of the IgM class, and that it appeared to be an anaemia- inducing factor. We have stimulated production of cold-active haemagglutinin in rats by injections of freeze-thawed autologous blood cells, but as yet have not seen evidence of anaemia in rats from this autoimmunization (unpublished). It appears that this rather simple test for cold-active haemagglutinin using compatible homologous erythrocytes treated with trypsin might be a useful indicator of suspected cases of autoimmune- 1ike blood disorders. SUMMARY Anaemia with splenomegaly and signs of glomerulonephritis were found associated with the late acute and post-acute phase of Trypanosoma lewisi infections of laboratory rats. The onset of the anaemia was associated with the peak of parasitaemia and the develop- ment of cold-active haemagglutinin (HA) for trypsinized rat erythro- cytes. It persisted with gradual recovery for as long as the trypano- somes and HA were detected in the blood. Signs of glomerulonephritis consisted of hypercellularity of the glomerular tuft, swelling of vascular endothelium and tubular epithelium, thickening of Bowman's membrane and tubular basement membrane, and abnormal numbers of hyaline casts in the distal convoluted tubules. Residual damage to the kidneys was not evaluated. 88 REFERENCES Cox, H. W., and G. C. Iturri. 1976. Autoimmune factors associated with anaemia in acute Haemobartonella and Eperythrozoon infections of rodents. Annals of Tropical Medicine and Parasitology_ 70:73-79. Cox, H. W., W. F. Schroeder, and M. Ristic. 1966. Hemagglutination and erythrophagocytosis associated with anemia of Plasmodium berghei infections of rats. Journal of Protozoology'13zsz7-332. Iturri, G. M., and H. W. Cox. 1969. Glomerulonephritis associ- ated with acute haemosporidian infection. Military Medicine (Special Issue) 134:1119-1128. Kibukamusoke, J. W., and M. S. R. Hutt. 1967. Histologic features of the nephritic syndrome associated with quartan malaria. Journal of Clinical Pathology_20:ll7-123. Luna, L. G. 1968. Manual of histological staining methods of the Armed Forces Institute of Pathology. 3rd Ed., McGraw- Hill Book Co., New York. Oki, Y., and K. Miura. 1970. Characteristics and roles of red cell antibodies in equine infectious anemia. Japanese Journal of Veterinary Research 32:217—227. Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Anaemia parasitaemia, erythrOphagocytosis and haemagglutinins in Babesia rodhaini infections. Annals of Tropical Medicine and Parasitology_60:3l-38. 10. 11. 12. 89 Soni, J. L., and H. W. Cox. 1974. Pathogenesis of acute avian malaria. I. Immunologic reactions associated with anemia, splenomegaly and nephritis of acute Plasmodium gallinaceum infections of chickens. American Journal of Tropical Medicine and Hygiene 23:577-585. Soni, J. L., and H. W. Cox. 1975. Pathogenesis of acute avian malaria. II. Anemia mediated by a cold-active autohemag- glutinin from the blood of chickens with acute Plasmodium gallinaceum infections. American Journal of Tropical Medicine and Hygiene 24:206-213. Soulsby, E. J. L. 1968. Helminths, arthropods and protozoa of domestic animals. 6th Ed., p. 573. The Williams and Wilkins Company, Baltimore. Thoongsuwan, S., and H. W. Cox. 1973. Antigenic variants of the haemosporidian parasite, Babesia rodhaini selected by Ig_vitro treatment with immune globulin. Annals of Tropical Medicine and Parasitology 67:373-385. Thoongsuwan, S., and H. W. Cox. 1977. Comparative studies of infectious anaemias in rats. 1. Haemolytic anaemia and glomerulonephritis associated with haemagglutinin in rats infected with a filterable agent. Annals of Tropical Medicine and Parasitology_(submitted). 90 Table l.--The means of spleen volumes, scores of kidney damage (SKD) and of the number of nuclei in the glomerular tuft (NGT) of 8 normal rats and 8 rats infected for 10 days with Trypanosoma lewisi. Infected Control Spleen volume 3.24 (2.8 - 3.7 ml) 1.0 (0.9 - 1.2 m1) (Range) SKD i S.E. 223.67 1 19.1* 47.00 i 3.06 NGT i S.E. 76.89 i 1.5* 52.43 i 3.81 *Students t test, significant at P > 0.01. 91 Fig. 1. --Mean and standard error of the mean (indicated by vertical lines) for erythrocyte counts (RBC x 106 ), trypanosome counts (trypanosomes x 105 ) and titers of cold- active haemagglutinin (HA titer) in rats after infection with l x 10 Trypanosoma lewisi parasites and in control rats injected with l x 1077 normal rat erythrocytes. Trypanosomes and HA were not found in blood of the control rats. RBC x 10‘/ mm3 - I l ,2 _ ”J H 92 H“ 0—0 RBC I. lew isi ATC infection O--O No. Tryp. I'I HA Titer Normal rots control A—A RBC ‘\\ \ C ‘\ “ ‘ “. l|llllllllllllllllllllllll 5 w I 0.6345 2" Figure 1 fi 8 P< 0.05.5mden1 Hes! Emu] /§0l X SEIWOSONVdAHl 2131M VH 93 Fig. 2.--Photomicrographs of sections of kidneys from rats infected for 10 days with Trypanosoma lewisi (A and B) and from a normal rat (C). In section A the glomerular tuft completely fills Bowman's space and the lumen of the adjacent convoluted tubules has been obliterated by swelling of the tubular epithelium; this nephron was given an SKD score of 4 plus. Approximately 100 nuclei may be counted in the glomerular tuft (NGT = 100). In contrast the nephrons seen in C received an SKD score of 0 and NGT count of approximately 45. Fibrinous adherence of the glomerular tuft to the thickened wall of Bowman's capsule. Thickening of the basement membrane of the tubules and some interstitial edema is evident (B). H and E stain 700 X. 94 Figure 2 Article 3 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS III. IMMUNOCONGLUTININ ASSOCIATED WITH NONSPECIFIC ACQUIRED RESISTANCE AND ANAEMIA Santi Thoongsuwan Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (To be submitted to Transactions of The Royal Society of Tropical Medicine and Hygiene, London, England) 95 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS III. IMMUNOCONGLUTININ ASSOCIATED WITH NONSPECIFIC ACQUIRED RESISTANCE AND ANAEMIAI’2 Santi Thoongsuwan3 Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 1This communication is from a thesis entitled "Comparative Studies of Infectious Anemias in Rats" submitted by the author in partial ful- fillment of the requirements for the Ph.D. degree from Michigan State University. His participation in these studies was made possible by a Faculty Fellowship award from the Chulalongkorn University, Bangkok, Thailand. 2This communication is Journal Article No. from the Michigan Agricultural Experiment Station. 3Present Address: Department of Microbiology Faculty of Pharmacy Chulalongkorn University Bangkok, Thailand 96 97 INTRODUCTION Past reports indicated that part of the resistance acquired by recovery from infectious anaemia was of a nonspecific nature. Rodents recovered from malaria were resistant to babesiosis and vice versa; mice with Eperythrozoon infections had an enhanced resistance to malaria, and ducks recovered from viral anaemia survived malarial infections that were lethal for controls (Cox, 1972; Cox and Milar, 1968; Ludford et al., 1969, 1972; Ott et al., 1967; Peters, 1965). Diseases from each of these infections are similar in that each is manifested by anaemia and splenomegaly which is accompanied by cold-active haemagglutinin (CAH) (Cox and Iturri, 1976; Cox et al., 1966; Schroeder et al., 1966). This syndrome of anaemia accompanied by autoantibody to erythrocytes has since been recorded for rats infected with a filterable agent and for rats with Trypanosoma lewisi infection (Thoongsuwan and Cox, 1977a, 1977b). Another autoantibody, immunoconglutinin (IK) has been associated with anaemia in malaria and trypanosomiasis (Woodruff, 1973). Coombs et a1. (1961) and Parappally et a1. (1973) pointed out that the conglutination activity of IX might have a role in nonspecific acquired resistance. Following a similar line of thought in discussing autoimmunity in malaria, Cox (1964) suggested that autoantibody possessing a detrimental effect on erythrocytes might be deleterious to plasmodial parasites and thus contribute to resistance. We have investigated this phenomenon of acquired nonspecific resistance associated with recovery from various infectious anaemias and have found it to be associated with IK. 98 MATERIALS AND METHODS Experimental animals and infections Male Sprague-Dawley rats weighing 100 to 180 gm obtained from Spartan Research Animals Inc., Haslett, Michigan, were used for all experiments. Animal care and experimental procedures were consistent with those promulgated by the Institute for Laboratory Animal Resources, National Research Council. The present studies were made using rat infectious anaemia (RIA) agent, the American Type Culture (ATC) strain of Trypanosoma lewisi, Babesia rodhaini and Plasmodium chabaudi. RIA agent is a filterable agent associated with a strain of I, lewisi maintained at this laboratory. Filtration experiments indicated that its diameter was smaller than 0.20 micron. While it has not been grown in cell culture, failure to detect other infectious forms led us to suspect that it is a virus. In mature rats RIA agent produces acute haemolytic anaemia, splenomegaly, glomerulonephritis and death within 8 to 10 days. The disease is less severe in young rats and those weighing 100 gm or less usually survive. Anaemia was accompanied by high titres of cold-active haemagglutinin (Thoongsuwan and Cox, 1977a). The ATC strain of I, lewisi was donated to the American Type Culture Collection by Dr. Robert G. Yaeger, Dept. of Tropical Medicine and Public Health, Tulane University, New Orleans, LA. We found that infections of rats with this strain caused a moderate anaemia with splenomegaly and nephritis which were accompanied by CAH. Our studies furnish no evidence that it was contaminated with RIA agent (Thoongsuwan and Cox, 1977b). 99 P, chabaudi was adapted to Sprague-Dawley rats by Musoke et a1. (1977). Its origin and methods for maintenance have been previously described (Cox and Milar, 1968). The origin and methods for maintain- ing B, rodhaini have also been described (Thoongsuwan and Cox, 1973). Standardized infections were induced by intraperitoneal injection of 1 m1 of a 1:100 dilution of blood from a rat infected 4 days previously with RIA agent, 1 m1 of a saline suspension of l x 106 trypanosomes from a rat infected for 5 days with I, lewisi, and 1 x 108 erythrocytes infected with B, rodhaini or P, chabaudi in 1 m1 saline. All experimental infections were standardized by described methods (Cox, 1957; Cox and Milar, 1968; Thoongsuwan and Cox, 1977a). Measurements of anaemia and_parasitaemia Blood obtained from the snipped tails of rats was drawn with a Sahli RBC hemocytometer pipette and diluted with Hayems solution. A Wrights stained blood film was prepared for determining the per- centage of parasitized erythrocytes of rats infected with I, chabaudi or B, rodhaini (Cox, 1957). Red blood cells and trypanosome counts were made microscopically in a Neubauer hemocytometer counting chamber. Estimates of viraemia with RIA were not determined. Autostimulation of immunoconglutinin in uninfected rats Kaolin that had been washed with distilled water was ground, autoclaved, and dried. Six ml of blood was drawn from each rat and the recovered serum of each was stored at -18 C. One ml of serum of each rat was mixed with 10 mg of kaolin and incubated at 37 C for 10 min. The mixtures were then washed once, resuspended to original 100 serum volume with 0.85% saline and each was mixed with an equal volume of Freund's complete adjuvant. A 1 ml injection was administered intramuscularly into each thigh of the donor rat. Two subsequent injections were given at weekly intervals using freshly prepared materials. One week after the last injection, they were bled for IK sera or infected for experimental purposes. Titration of immunoconglutinin (IK) IK from blood of experimental and control rats was titred by its conglutination of complement-fixed sensitized sheep red blood cells (SRBC) as described by Coombs et a1. (1961) except that rats were used to generate anti-SRBC serum. Cooke microtiter methods were used to test serum for IK activity. Rats were given a single intraperitoneal injection of 1 ml of 2% suspension of washed SRBC and 9 days afterward the serum was recovered from blood drawn by cardiac puncture under ether anesthesia. The pooled serum was stored at ~18 C. Optimal concentration of anti-SRBC giving least haemolysis and strong agglutination with IK positive serum was predetermined. Wells of microtiter plates were charged with 0.025 ml of 2-fold diluted sample, 0.025 ml of 1:10 heat inactivated horse serum, 0.025 ml of 0.5% complement-fixed sensitized SRBC and 0.05 ml of saline in that order. Negative controls for each sample consisted of sensitized SRBC treated with heat inactivated horse serum. The plates were covered with adhesive plastic film and mechanically shaken for 1 min before incubation at 37 C for 30 min. Final readings of the conglutination titre were made after storage overnight at 4 C. 101 Tests for cold-active haemagqutinin (CAH) CAH was detected in plasma or serum with trypsinized rat erythrocytes as previously described (Thoongsuwan and Cox, 1973). Tests for anaemia, nonspecific acquired resistance, CAH and 1K in rats recovered from RIA, malaria, babesiosis and trypanosomiasis Since blood induced infections were used in these experiments, control animals were injected intraperitoneally with l x 109 washed rat erythrocytes. Rats recovered from RIA had been infected when young (100 gm) and recovery of anaemia was complete 4 weeks later (Thoongsuwan and Cox, 1977a). Rats infected with I, 122121 were not completely recovered from anaemia until 30 days after infection (Thoongsuwan and Cox, 1977b). Recovery of rats from B, rodhaini infection was facili- tated by an intraperitoneal injection of 1 m1 of 0.5% acriflavine hydrochloride 4 days after infection and recovery from anaemia was complete 3 weeks later. Rats recovered from RIA and B, rodhaini infections were each given a second injection of 1 ml of whole blood from rats with acute RIA or from rats with acute babesiosis. They were challenged ten days later. Twenty-five RIA recovered and 25 control rats were challenged with B, chabaudi. Groups of 25 recovered and 25 control rats were challenged with B, rodhaini. Groups of 6 recovered and 6 control rats were challenged with I, 122121: Challenge with RIA agent was made in 18 rats recovered from B, rodhaini and 16 recovered from I, lewisi, using a single control group of 16 rats. 102 Blood from 6 control and 6 experimental rats for RBC counts and staining was obtained daily commencing one day before challenge. Four animals of each group served as a source for plasma to be tested for CAH and IK activity. Samples of 3 ml were drawn on alternate days by cardiac puncture into 0.3 ml of heparinized saline. Mortality was recorded daily for each group. Tests for anaemia, nonspecific acquired resistance, CAH and IK in rats autostimulated by injections with autologous serum absorbed on kaolin Twelve normal rats were bled for serum samples before they were injected with autologous serum absorbed on kaolin as described. After autostimulation, blood for RBC counts and staining was taken on 6 experimental and 6 control rats. Blood was drawn for CAH and IK assays from 4 of the rats in each group. All rats were given 1 x 107 B, rodhaini infected rat blood cells the following day. RBC counts and blood films were taken daily and blood to be tested for CAH and IK activity was taken at 2 day intervals. Mortality for each group was recorded. EXPERIMENTAL RESULTS The relationships of immunoconglutinin and cold-active haemagqutinin in serum of rats with acute rat infectious anaemia (RIA), trypanosomiasis, babesiosis and malaria and in serum from rats recovered from each infection Pools of serum taken from rats during acute infection and after recovery were tested for 1K and CAH (Table l). Rats with acute 103 infections showed titres of IX ranging from 640 to 5120 compared to 20 in normal rat serum. After recovery IK titres ranged from 80 to 640. CAH was found in the serum pools from rats with acute infections but was not detected in serum of recovered rats. Anaemia, immunoconglutinin, cold-active haemagqutinin and survival from RIA agent infection in rats recovered from T. lewisi and B. rodhaini infections Rats recovered from I, lewisi and B, rodhaini were fully mature on day 30. Results of RBC counts, IX and CAH titres, and mortality are presented in Table 2. Anaemia was evident l to 2 days earlier in rats recovered from I, lewisi and B, rodhaini infections than in control rats. Red cell counts fell more precipitously and were lower in the control animals than in recovered rats. This early anaemia was associated with a higher initial and a more rapid increase in IK titres after challenge than seen in the control rats. CAH was found in each group of chal- 1enged rats, but titres did not differ in the three groups. Fourteen of 18 rats recovered from B, rodhaini and 13 of 16 recovered from I, lewisi infections survived RIA agent infection and appeared to be normal 3 weeks later. None of the control rats were alive after day 9 and all exhibited haemaglobinuria at the time of death. Evidence of recrudescent B, rodhaini or I, lewisi infection was not found. 104 Protection from acute malaria and babesiosis in rats that had recovered from RIA This experiment was performed with young rats (80-100 gm). These animals exhibited only moderate signs of anaemia with no haemoglobinuria following the first inoculation of RIA agent and less anaemia following the second injection. Data on parasitaemia, RBC counts, IK and CAH activity on these and control rats after challenge with B, rodhaini and B, chabaudi are presented in Tables 3 and 4. After challenges, signs of anaemia developed earlier in the recovered rats than in the controls. The early reductions in RBC counts were accompanied by lower B, chabaudi and B, rodhaini para- sitaemia. Early anaemia and reduced parasitaemia were associated with higher initial and more rapid increases in IK titres. CAH titres of control and experimental rats did not differ. When anaemia developed in the control rats the RBC counts fell more precipitously and remained lower than those observed in recovered rats. Thirteen of 25 rats challenged with B, rodhaini and 12 of 25 infected with B, chabaudi survived and appeared normal 3 weeks after infection. All 25 control rats for each infection died within 8 days. Protection from trypanosomiasis and anaemia in rats recovered from acute RIA The RBC and trypanosome counts from 6 RIA recovered rats and 6 control rats after challenge with I, IEBI§I_are presented in Table 5. Additional rats for CAH and IK testing were not available. In both groups infections became patent and did not differ until day 5 when the trypanosome counts of the recovered rats were significantly lower 105 than those seen in control rats. On the previous day the erythrocyte counts of the recovered rats were significantly lower than those of the controls. The trypanosomal counts of the recovered rats remained lower and reverted to negative earlier than those of the control group. The lower trypanosome counts of recovered animals were accompanied by lower erythrocyte counts from the 4th through the 16th day. There- after, the RIA rats recovered from anaemia more rapidly than did the controls. Protection from babesiosis in rats with autostimulated immunoconglutinin The RBC and parasitaemia counts of rats infected with l x 107 B, rodhaini infected cells are shown in Table 6. Both experimental and control rats developed parasitaemia at equal rates until the 5th day when both anaemia and reductions in parasitaemia became evident in the experimental group. The experimental rats were clear of para- sitaemia 2 days earlier than the controls. Fifty percent of the stimulated rats and 25% of the control rats survived the challenging infection. On day 0 the stimulated rats had mean titres of 160 for 1K and 117 for CAH. Throughout the experiment these titres rose more rapidly and were higher than those found in the controls. DISCUSSION These experiments confirmed and extended previous observations on nonspecific acquired resistance associated with recovery from anaemia-inducing infections. Enhanced resistance to Plasmodium berghei and to B, chabaudi has been associated with Eperythrozoon coccoides 106 infections (Ott et al., 1967; Peters, 1965). The resistance of rodents recovered from malaria to babesiosis, and vice versa, observed by Cox and Milar (1968) was confirmed (Cox, 1972). Ludford et a1. (1969, 1972) demonstrated that ducks recovered from viral anaemia had enhanced resistance to malaria. The present work has shown that this nonspecific acquired resistance can be stimulated in rats by infections of RIA agent and by I, lewisi. Further, it was shown that the resistance was functional against RIA agent and trypanosomal infections as well as against malaria and babesiosis. The resistance was manifested as a reduction in parasitaemia which was accompanied by significant reductions in erythrocyte counts manifested on the 2nd or 3rd day after infection. The reduction in parasitaemia and early anaemia were associated with titres of IK that were higher than those seen in the control animals. The association of IK with nonspecific acquired resistance was further emphasized by the resistance of uninfected rats with autostimulated IK to B, rodhaini challenge. This resistance was temporally associated with CAH, which was found in blood of rats during the course of each infection and in blood of uninfected rats with autostimulated IK. It was therefore not clear whether this resistance could be attributed to IK alone. CAH was associated with acute Babesia, Trypanosoma, Plasmodium, Haemobartonella and RIA agent infections (Cox and Iturri, 1976; Cox et al., 1966; Schroeder et al., 1966; Thoongsuwan and Cox, 1973; 1977a; 1977b). In the present experiments CAH was stimulated in unin- fected rats simultaneously with IK by injections of kaolin adsorbed 107 rat serum in Freund's complete adjuvant. CAH had been stimulated in normal chickens by injection of disrupted autologous erythrocytes and was detected in chicken plasma with trypsinized human type "0" erythrocytes (Soni and Cox, 1975a). We stimulated CAH in rats by injections of disrupted autologous erythrocytes and demonstrated a reaction of the antibody with rat erythrocyte stroma (unpublished). CAH is therefore considered to be autoantibody to the erythrocyte stroma antigens (Friedenreich, 1928). IK is autoantibody to fixed C3 and/or C4 (Lachmann, 1966; 1967; Lachmann and Muller-Eberhard, 1968). It has been associated with a number of infectious diseases involving anaemia and splenomegaly in man (Woodruff, 1973). We found IK associated with anaemia and splenomegaly from infections of RIA agent, I, lewisi, B, chabaudi and B, rodhaini infections in rats. It is of interest that Woodruff (1973) speaks of the "anaemia- big spleen syndrome" associated with IK in infectious diseases, and Bg_speak of a syndrome of anaemia, splenomegaly and nephritis associ- ated with CAH as the "infectious anaemia syndrome" (Cox and Iturri, 1976). From the present experiments it would appear that Professor Woodruff's and our syndrome may be the same. Although the nature of the factors stimulating 1K in these infections is unknown it seems reasonable to assume that it was stimulated by complement fixing immune complexes. However, such complexes were not sought in the present study. Candidates for 1K stimulation might be a complex of stroma antigen and CAH. Parasite antigen and its antibody may also be present as soluble complex in 108 the blood during acute infectious anaemias (Musoke et al., 1977; Soni and Cox, 1975a). Soni and Cox (1975b; 1975c) demonstrated that soluble complexes of soluble serum antigen (SA) and its antibody (ABSA) were present in the blood of malarious chickens, and that the com- plexes caused anaemia and nephritis when injected into normal birds. The presence of SA and ABSA was associated with anaemia in rats resulting from malaria, babesiosis and haemobartonellosis (Cox and Iturri, 1976; Cox et al., 1968; Soni and Cox, 1974; Thoongsuwan and Cox, 1973). Other factors may have formed immune complexes that fixed complement. Barrett-Connor (1967) reported a case of Plasmodium yiyag malarial anaemia associated with positive tests for Wassermann antibody and for rheumatoid factor. We found Wassermann antibody and CAH associated with anaemia and glomerulonephritis in rodent babesiosis (Iturri and Cox, 1969). Complexes of any of these antibodies with their respective antigens could serve to stimulate IK. While it is clear that IK was associated with this phenomenon of early anaemia and enhanced resistance, the presence of other immune bodies makes it unwise to suggest that IK alone contributed to resis- tance. Anaemia resulted from injections of SA into normal animals, and afterwards they had an enhanced resistance to heterologous species and genera of haemosporidian parasites (Cox, 1966; Sibinovic et al., 1967; Corwin and Cox, 1969). From the suggestion of Dixon (1966) we suspect that soluble complexes of SA and ABSA may have become bound to erythrocytes and/or parasites to mimic opsonin and cause these particles to be sequestered and phagocytized. Coombs et a1. (1961) presented evidence that the conglutinating activity of IK with 109 complement-fixed complexes of bacteria and antibody would enhance the clearing of bacteria and thus serve as a factor in nonspecific acquired resistance. It is possible that IK may play a similar role involving erythrocytes and parasites that have been coated with antigen-antibody complexes. SUMMARY Rats recovered from infectious anaemias had an acquired non- specific resistance. Recovery from trypanosomal and babesial infec- tions enhanced the resistance to infections with filterable rat infectious anaemia (RIA) agent, and recovery from RIA made rats more resistant to plasmodial, babesial and trypanosomal infections. The resistance was manifested by reduced parasitaemia which usually became evident on the 2nd or 3rd day of infection and which was accompanied by significant anaemia. Mortality from challenge of the recovered rats was usually less than those of the controls. Immunoconglutinin (IK) was detected in high titres in animals during acute stages of infection and remained present in lower amounts after recovery. After the recovered rats were challenged with a heterologous agent, the IK titres became elevated earlier and were usually higher than those of the controls. Uninfected rats with autostimulated IK also exhibited similar enhanced resistance to challenge. However, infections and autostimulation of IK also stimulated production of cold-active haemagglutinin (CAH). It was therefore not clear that the resistance could be attributed to IK alone. 110 REFERENCES Barrett-Connor, E. 1967. Amer. B, Trop. Med. Hyg. 16:699-703. Coombs, R. R. A., A. M. Coombs, and D. G. Ingram. 1961. The serology of conglutination and its relation to disease. Blackwell, Oxford. Corwin, R. M., and H. W. Cox. 1969. Mil. Med. (Suppl.) 134:1258-1265. Cox, F. E. G. 1972. Parasitol. 65:379-387. Cox, H. W. 1957. B, Immunol. 79:450-454. Cox, H. W. 1966. Mil. Med. (Suppl.) 131:1195-1200. Cox, H. W., and G. C. Iturri. 1976. Ann. Trop, Med. Parasitol. 70:73-79. Cox, H. ., and R. Milar. 1968. Amer. B, Trop. Med. Hyg. 17:173-179. W Cox, H. W., R. Milar, and S. Patterson. 1968. Amer. g, Trop. Med. Hyg. 17:15-18. Cox, H. W., W. F. Schroeder, and M. Ristic. 1966. B, Protozool. 13:327-332. Dixon, F. J. 1966. Mil. Med. (Suppl.) 131:1233-1234. Friedenreich, V. 1928. Acta. Path. Microbiol. (Scand.) 5:59-101. Iturri, G. M., and H. W. Cox. 1969. Mil. Med. (Suppl.) 134:1195-1200. Lachmann, P. J. 1966. Immunol. 11:263-271. Lachmann, P. J. 1967. Advanc. Immunol. 6:479-527. Lachmann, P. J., and H. J. Muller-Eberhard. 1968. B, Immunol. 100:691-698. Ludford, C. G., R. M. Corwin, H. W. Cox, and T. A. Sheldon. 1969. Mil. Med. (Suppl.) 134:1276-1283. Ludford, C. G., H. G. Purchase, and H. W. Cox. 1972. Exp, Parasitol. 31:29-38. Musoke, A. J., H. W. Cox, and J. F. Williams. 1977. Amer. B, Trop. Med. yg. (in press). Ott, K. J., J. K. Austin, and L. A. Stauber. 1967. Exp, Parasitol. 21:68-77. 111 Parapally, N. P., and D. G. Ingram. 1973. Immunology 25:523-530. Peters, W. 1965. Exp. Parasitol. 16:158-166. Schroeder, W. F., H. W. Cox, and M. Ristic. 1966. Ann. Trop. Med. Parasitol. 60:31-38. Sibinovic, K. H., S. Sibinovic, M. Ristic, and H. W. Cox. 1967. B, Parasitol. 63:327-336. Soni, J. L., and H. W. Cox. 1974. Amer. B, Trop. Med. Hyg. 23:577-585. Soni, J. L., and H. W. Cox. 1975. Amer. B, Trop. Med Hyg 24:206-213. Soni, J. L., and H. W. Cox. 1975. Amer. 24:423-430. |La l': H O I: I??? Soni, J. L., and H. W. Cox. 1975. Amer. 24:431-438. “a li la? Thoongsuwan, S., and H. W. Cox. 1973. Ann. Trop. Med. Parasitol. 67:373-385. Thoongsuwan, S., and H. W. Cox. 1977a. Ann. Trop, Med. Parasitol. (submitted). Thoongsuwan, S., and H. W. Cox. 1977b. Ann. Trop. Med. Parasitol. (submitted). Woodruff, A. W. 1973. Trans. B, Soc. Trop. Med. Hyg. 67(2):313-328. 112 Table l.--Immunocong1utinin (IK) and cold-active haemagglutinin (CAH) titres of pooled serum from 5 rats with acute and 5 recovered rats infected with rat infectious anaemia (RIA) agent, Babesia rodhaini, Plasmodium chabaudi and Trypanosoma lewisi. Infection IK CAH Acute Recovered Acute Recovered RIA 5120 320 512 0 Babesia rodhaini 2560 640 256 0 Plasmodium chabaudi 5120 80 256 0 Trypanosoma lewisi 640 80 128 0 Normal rats 20 0 113 mo.o H oH.v Hv.o H oo.m HH HnH owvm nw.o H um.v HRH mva mm.o H ov.m oH mm.o H mm.m nv.o H Ho.v m mHN comm wn.o H mm.m mmm mmm om.o H oH.v w mm.o H Hm.m 5v.c H mv.m n mmN own 111mo.o H wn.m va mom «10v.o H mm.m cow moo «mm.o H mm.N o mm.o H vH.v mm.o H mm.m mn.o H ww.m m mvH owe mm.o H on.m MHN oNH NN.o H Ho.h HnH mH mm.o H mm.» v mH.o H mH.n 0H.o H Hw.h mH.o H ~5.w m cm mmH an.o H Nn.n mm ow mH.o H ve.w mu mH aH.o H Hm.» N NH.o H Hn.w NH.o H mo.w wH.o H ov.w H o mmH NH.o H 5N.w 0 mm mm.o H mm.w 0 NH mH.o H Hv.w o zOUOH mvmm EOHM UOH0>OUOH mud“ mama Houpzou .Hcowm HHEomcm msoHuoomnH any :qu owcoHHmzo Houmm Hchcvou mHmonmm can HmHon «semocmmxuh mo mcoHuoomaH aonm wouo>ooon .muaa mo moHuHu ewes .H.m.m H 0 OH x ummv m Houucoo macaw quHmuuoE van H=HpomuvHoo van HxHV :HnHusHmcooocsaaH as you mucsoo ouxoousuxno we wanna vumvcwum H :w0211.m oHan 114 .Hamop u acoeaumv HH6>Huoommou No.o v a 6:6 Hoo.o A a pm Hemomeeme 1.6 .66>H>H=m meek as 61“ mo am.aa.4. .vo>H>Hsm mama 0H onu mo me.Hw«a .n xmv :o HovchEoH ecu .o xmv no voHv mama oH on» we anon; 0 mm Hm.o H Hm.n o no mH.o H nm.w on mm.o H wv.n NH.o H v~.w wH v mam om.o H nv.o m own m~.o H mo.n 0H mm mmNH hm.o H ~w.m noH mmvH n~.o H om.n vH Ho.o H «H.m w~.o H ou.o mH noH owNH mm.o H mo.m HAH mmHN om.o H mm.o NH .m.m H .m.m H .m.m H =OUQH w “my. scum cono>ooon muam mama Houucou .eoseHueou--.N oflame 115 o mH.o H cm.v NH mm mmHN c nm.o H no.n oH + mm.o H hm.~ m Hem ovmm om.o H on.w m~.o H ~m.~ m «amH.m H vn.Hm 11mm.o H mo.m A omm ommH mww.v H ov.mm mm.o H mH.m «mHN Home «NN.H H oo.mo ¢m~.o H mv.m c awn.m H mm.w~ nom.o H m~.m mH.N H mm.wm wm.o H mv.n m mvH nwm eve.~ H vw.m eoH.o H om.n mm noH ew.~ H mH.n~ mo.o H mH.m v am~.o H ww.H eHH.o H ww.n mv.c H wn.m NH.o H Hm.» n mu no omo.o H oc.o ono.o H om.w mm m nH.o H HH.H HH.c H ow.m N Ho.o H HH.o wo.o H mm.m No.0 H mH.o oH.o H nn.m H o noH o ao.o H mm.w 0 MH o nH.o H mm.m o :5 a: .m.m H E 3. bohmwmumm :5 a: .m.m H ma 3. powwow. Mmmwumwum ooou mHHMI, mama Houucou \ .Hchnvou «Hmonmm :HHz omcoHHmno Houma Hooou m~ use many Houuaoo mm macaw AHHHHHHOE nae HzHuum uuHoo use HxHV :HcHuuscoooasaeH mo moHuHu came .H.m.m H mm Hg mouxoonsuxno vouHuHmanum mo ommucoouom any .H.m.m H ooH x ummv mes Hon muczoo ouxuonnuxno mo uouno Humvcmum H cmozuu.n oHnee 116 .Humou H Humv3Hmv >Ho>HHoommoH mo.o v a wzm Ho.o v a .Hoo.o v a He HeaonwemHmo.n.m .H6>H>H=H Hmm--a 166 H1 HHHHHHHoa Hw44. .5 H66 an HHHHHHHoa Hood. o mmH o mH.o H ou.w em o NH.o H on.» mm o ov.o H Nu.n ON 0 00H 0 c~.o H v~.n wH o mm.o H om.o oH o How o mH.o H no.6 HH :OUOH mumm mumm HOHHGOU V .eeaeHaeou--.m HHBHH 117 o o~.o H Hm.m NH Hm oewm .H.+ H~.o H HH.H oH mm.o H oo.H a~.o H mm.» m .4HNH ..o~Hm ..-.H H oo.oH ..o~.o H HH.~ m m~.~ H oo.Hn H~.o H ~H.~ A AHN omen HHH.H H om.HH Nn.o H mm.m .AHN 406m Ham.” H oo.mo .mm.o H om.~ 6 «HA.» H HH.m~ Hm~.o H mH.m HH.~ H mh.mo 6N.o H m6.m m omw NOAH HAH.H H HH.HH HoH.c H om.“ HAH Hmw HN.H H mm.om oH.o H HH.H H HNH.o H mo.~ HHH.o H 6H.H m~.o H am.e wc.o H Ho.m H m- AHH HH.o H 55.0 HH.o H we.» mm oH HH.o H m~.H HH.o H mm.» N Ho.o H HH.c HH.o H 66.» No.o H HH.o HH.o H cm.» H o A-H o NH.° H me.” o A o oH.o H Hm.» o :«u HH .m.m H ma H acmmwmummlu :ooou muwm mudm Houpcou .Hvsmnmsu asHvoEmen nqu omcoHHmno Houma Hooon mN vca mama Houucou mm macaw HHHHHHHoa us“ H=HuomunHoo vcm HxHV =H:HH=Hw:ooo:=eaH mo moHHHH :woa .H.m.m H mm Hg mouxuonspxuo voNHHHmmumm * .H.m.m H ooH x ummv nee Hon mpcaou ouxoounuxuo we wanna vuwvcwum H :woz--.v «Hawk 118 .HHHoH H Heoesumv Hoo.o v a HH HanuHchmHmH .oHnuucsouaa Han o>HuHmom1HH .vo>H>H:m wmv11w xmu A: xHHkuHoa ammHH .A H66 an HHHHHHHoe Hoofi. o HoH o o~.o H 46.x Hm o mH.o H mm.” NH o AH.o H HN.A on o HHN o AH.o H o~.H HH 0 AN.o H mm.e 6H o mmw o -.o H w~.6 HH =oueu mumm. mum: Houunou .eoscHanou--.H OHHHH Table S.--Mean number of red blood cells per mm3 number of trypanosomes per mm3 (T X 10 11 9 5 (RBC x 106), mean ) and standard error of the mean (S.E.) in blood of 6 control rats (Control) and 6 rats recovered from rat infectious anaemia (RIA-Recovered) after challenge with Trypanosoma lewisi. Days 0 RBC x 10 1 S.E. T x 10 1 S.E. ontrol RIA-Recovered Control RIA-Recovered 0 9.02 1 0.10 .93 1 0.11 -* - l 9.06 t 0.06 .43 1 0.10 +** +** 2 9.03 1 0.09 .01 1 0.07 0.06 1 0.01 .02 1 0.01 3 9.12 1 0.15 .65 1 0.19 0.45 1 0.05 .31 1 0.07 4 8.52 1 0.14 .68 1 0.10a 2.14 1 0.24 .44 1 0.38 5 7.38 1 0.15 .11 1 0.18 5.62 1 0.80 .06 1 0.80d 6 6.98 1 0.26 .48 1 0.31 6.64 1 0.49 .91 1 1.12d 7 6.90 1 0.18 .46 1 0.13 7.10 1 0.49 .61 1 1.26d 8 6.53 1 0.17 .23 1 0.15 6.53 1 0.48 .82 1 1.11d 9 7.00 1 0.28 .19 1 0.19 6.63 1 0.72 .77 1 0.86b 10 6.98 1 0.28 .13 1 0.31 5.90 1 0.79 .37 1 0.61a 12 6.97 1 0.20 .50 1 0.42 5.23 1 0.59 .87 1 0.433 14 7.08 1 0.23 .93 1 0.47 3.92 1 0.61 .85 1 0.42b 16 6.99 1 0.28 .38 1 0.39 3.90 1 0.69 .44 1 0.30a 18 7.40 1 0.19 .01 1 0.33 2.36 1 0.58 - 20 7.18 1 0.27 .59 1 0.143 1.27 1 0.64 - 24 7.67 1 0.28 .31 1 0.12a 0.49 1 0.42 - 30 8.79 1 0.15 .23 1 0.07 - - *No trypanosome found. a,b,c,d **Rare trypanosomes found. Significant at P < 0.001, P < 0.01, P < 0.02 and P < 0.05 respectively (Student t test). .wouoouov Ho: m6: :HHoom66H Hoo.o v a 6:6 .m.wH ma: xH>H=m won .w Haw co HOHv macaw mH:H cH mama NH mo use meam .vo>H>H:m me .w xwv :o uoHu macaw mHnu =H mama NH we use 2521 120 0.0m n.00H 1 0N.0 H «0.5 h.Nv 0.00H 1 50.0 H 00.0 QH 1 00.0 H 00.0 1 0v.0 H 00.? NH HRH 0.000 1 M00.0 H 0H.m b.00H 0.000 + 00.0 H BH.M 0H D1 m00.0 H Hn.¢ Nh.0 H mn.N 00.0 H 00.N 0 b.0Nv 0.000 U00.H H mh.H «#vm.0 H 00.0 m.va 5.000H 0h.N H 0N.0H 10v.0 H QV.N 0 mH0.0 H m0.vN 00.0 H 5N.v <0.m H 0h.Nv 50.0 H MN.V h m.va 0.00mN hN.M H 0N.0H «Hm.0 H H0.0 NON b.005H 50.0 H 00.0N 0N.0 H H0.h 0 h0.H H 00.? 0H.0 H H0.0 m0.H H 00.0 HN.0 H H0.0 m 0.0HN n.00H 0H.0 H MN.H 0H.0 H N0.0 0.50H 0.0V Nn.0 H MN.H ¢H.0 H 00.0 v 50.0 H Nm.0 0H.0 H 00.0 00.0 H v0.0 0H.0 H mv.0 m 5.05H 0.00H No.0 H 00.0 NN.0 H 00.0 h.Vh 0.00 N0.0 H v0.0 0H.0 H 00.0 N + 0H.0 H 05.0 + 0N.0 H 0v.0 H 0.5HH 0.00H 1 0H.0 H 00.0 0 m.nN 1 mN.0 H Hm.0 0 =HHum1vHoo use Hva :HcHusHmsooocseaH mo mouHHu cues .Hmm *0 mouxoounuxuo HONHHHmmHmm mo ommusoouom .HooH x umm0 nae Hon mHHoo vooHn wen we H.m.m0 name map mo Houuo vnmvcapm H :60211.o oHnme Article 4 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS IV. STUDIES OF THE NATURE OF IMMUNOCONGLUTININ ASSOCIATED WITH INFECTIOUS ANAEMIA OF RATS AND ITS ROLE IN NONSPECIFIC ACQUIRED RESISTANCE Santi Thoongsuwan Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 (To be submitted to Transactions of The Royal Society of Tropical Medicine and Hygiene, London, England) 121 COMPARATIVE STUDIES OF INFECTIOUS ANAEMIAS IN RATS IV. STUDIES OF THE NATURE OF IMMUNOCONGLUTININ ASSOCIATED WITH INFECTIOUS ANAEMIA OF RATS AND ITS ROLE IN NONSPECIFIC ACQUIRED RESISTANCEI’Z Santi Thoongsuwan3 Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 1This communication is from a thesis entitled "Comparative Studies of Infectious Anemias in Rats" submitted by the author in partial ful- fillment of the requirements for the Ph.D. degree from Michigan State University. His participation in these studies was made possible by a Faculty Fellowship award from the Chulalongkorn University, Bangkok, Thailand. 2This communication is Journal Article No. from the Michigan Agricultural Experiment Station. 3Present Address: Department of Microbiology Faculty of Pharmacy Chulalongkorn University Bangkok, Thailand 122 123 INTRODUCTION Thoongsuwan et a1. (1977) reported that immunoconglutinin (IK) was associated with anaemia in rats infected with the filterable agent of rat infectious anaemia (RIA), Trypanosoma lewisi, Babesia rodhaini, and Plasmodium chabaudi. Rats that had recovered from each of the infections maintained reduced but significant titres of this autoantibody. When the recovered rats were challenged with an agent other than the one used for initial infection, they exhibited a non- specific acquired resistance to the heterologous infection. This resistance was manifested 2 to 3 days after infection by significant reductions in parasitaemia which was accompanied by significant reductions in the erythrocyte counts. The recovered rats also had less mortality after challenge than the controls. After challenges, the IK titres of the recovered rats became elevated earlier and were generally higher than those of the controls. However, a role for IK in this resistance was unclear. Antibody to erythrocytes in the form of cold-active haemagglutinin (CAH) was also generated during the infections. An additional factor may have been involved. Soluble serum antigen (SA) and its antibody (ABSA) have been associated with anaemia and acquired resistance of unrelated infectious agents (Cox, 1966; Sibinovic et al., 1967, 1969; Cox et al., 1968; Cox and Iturri, 1967). Soni and Cox (1974, 1975a, 1975b, 1975c) demonstrated that CAH and complexes of SA and ABSA may have a causal relationship in anaemia and nephritis. Thus, factors other than IK may have contributed to the resistance. 124 We present the results of experiments designed to ascertain whether IK and/or ABSA contributed to the nonspecific acquired resistance of infectious anaemia. MATERIALS AND METHODS Experimental animals Male Sprague-Dawley rats and white Swiss mice were obtained from Spartan Research Animals Inc., Haslett, Michigan. Methods for animal care and the experimental procedures were consistent with those promulgated by the National Institute for Laboratory Animal Resources of the National Research Council. Experimental infections Four taxonomically unrelated agents of infectious anaemia were used. The filterable agent of RIA was discovered and proven to cause infectious anaemia of rats at this laboratory (Thoongsuwan and Cox, 1977a). The American Type Culture (ATC) strain of'I, lewisi was described (Thoongsuwan and Cox, 1977b). The E, rodhaini and E, chabaudi infections in rats were described by Thoongsuwan and Cox (1973) and Musoke et a1. (1977). Standardized infections for each agent were prepared as described (Cox, 1957; Thoongsuwan and Cox, 1977a). Preparation of serum fractions Rats infected with g, rodhaini for 4-5 days were exsanguinated by cardiac puncture under ether anaesthesia. Serum was recovered 125 from the clotted blood after setting overnight at 4 C and then stored at -18 C until used. Rats that had recovered from g, rodhaini infection were hyper- immunized by 3 weekly intraperitoneal injections of 1 ml of whole heparinized blood from rats heavily infected with E, rodhaini. One week after the final injection the rats were exsanguinated by cardiac puncture under ether anaesthesia. Serum was recovered and stored as just described. Serum collected from blood of rats with acute infection and from hyperimmunized rats was treated 3 to 4 times with ammonium sulfate at 50% saturation (50% SAS) until the precipitate was clear of haemoglobin discolouration. The recovered globulin was dialyzed at 4 C with repeated changes of borate buffered saline, pH 8.4, 0.175 ionic strength, until sulfate ion could no longer be detected with barium chloride solution. Each preparation was adjusted to its original serum volume. Tests of the globulin fraction with trypsinized rat erythro- cytes was performed as described by Thoongsuwan and Cox (1973) revealed the presence of CAH in globulin of rats with acute infection. CAH was removed by repeated absorptions at 4 C with trypsinized rat erythrocytes as described (Soni and Cox, 1974). The globulin fraction was tested for IR using methods modified (Thoongsuwan et al., 1977) from those of Coombs et al. (1961). ABSA was detected with SA from serum of rats with acute P, chabaudi or E, rodhaini infections by immunodiffusion in gel plates obtained from Research Products Division, Miles Laboratories Inc., Kankakee, Illinois. 126 The globulin fraction was pre-equilibrated with eluting buffer and subjected to gel filtration on Sephadex GZOO column at 4 C using a gel bed of 5 x 90 cm and a flow rate of 24 ml/hr. The column was pre-calibrated using 2 ml of 1% solution of blue dextran as the marker for 198 peak. The globulin containing 26.5 mg/ml of protein was constituted to 3% sucrose and a 10 ml sample was underlayed onto the column. Fractions were eluted with 0.10 M pH 7.5 phosphate buffer in 0.85% NaCl solution and collected in 4 ml aliquots. Protein concen- tration was automatically monitored at 280 nm. Samples were stored at 4 C until used. Pools of the 19$, 73, and 45 fractions were concentrated in an Amicon untra filtration unit on a UM-lO membrane. After dialysis overnight in 0.01 M pH 7.5 phosphate buffer in 0.85% NaCl (PBS), the final protein concentration of each pool was determined as described (Lowry et al., 1951) and adjusted to 6 mg/ml for acute and 9 mg/ml for hyperimmune globulins. The samples were tested for ABSA with serum from rats with acute B, rodhaini and P, chabaudi infections. Studies of the nature of IR stimulated in rats during B. rodhaini infection A portion of the concentrated fraction pool that contained IK activity was treated with Z-mercaptoethanol (2-ME) as described (Chan and Deutsch, 1960). Treated and untreated portions were tested for IK reactivity against sheep erythrocytes sensitized with antibody (EA) that had been fixed with C14 (EAC14) and with C143 (EAC143) fragments of human complement. The EAC14 intermediate was prepared with optimally sensitized EA just as described by Linscott (1975). Ill-F. I’ll! III 1 127 The EACl43 intermediate was prepared from EAC14 intermediate with C2 that was purified following methods of Dias da Silva and Lepow (1967), and with C3 prepared by methods of Nilsson and Mfiller- Eberhard (1965). A suspension was made of 1.5 x 108 EAC14 cells/ml in sucrose-veronal buffered saline of 0.065 ionic strength, this 0.065 u buffer was prepared as described by Rapp and Borsos (1963). Five m1 of the suspension were reacted with 5 m1 of C2 (50 effective molecules/ml) for 20 min at 30 C. The resultant EAC142 was washed 3 times with 0.065 u buffer and adjusted to S x 108 cells/m1. One ml of EACl42 was mixed with 1 ml of C3 (1 mg/ml) and incubated for 90 min at 37 C to decay functional C2 activity, the preparation was then washed. The resulting EAC143, and EAC14 described above, were held at 0 C until tested for conglutinating activity with IK in Cooke microtiter plates as described (Thoongsuwan et al., 1977). Fluorescein isothiocyanate (FITC) conjugation ofglobulin fractions Globulin of rats containing IK stimulated by injections of autologous normal serum absorbed on kaolin as described by Thoongsuwan et a1. (1977) was absorbed free of CAH as described (Soni and Cox, 1974). The 75 fraction of CAH-free globulin from rats hyperimmunized by B, rodhaini infections, containing ABSA and no IK, was prepared as described above. These preparations were conjugated with FITC, absorbed once with animal charcoal, and once with washed buffy coat cells of normal rats until unreactive with blood films or spleen impression slides of normal rats (Goldman, 1968; Thoongsuwan and Cox, 1977a). 128 Blood films and spleen impression slides prepared from rats with acute RIA, trypanosomiasis, babesiosis, malaria, and from normal rats were fixed with absolute methanol. They were incubated with a 1:10 dilution of the FITC conjugates at 37 C for 30 min, rinsed twice with PBS, and a coverglass was mounted with 90% glycerine in PBS. Fluorescent activity was studied with a Zeiss Fluoroscope equipped with an Osram HBO mercury lamp. Photomicrographs of the reactions were made with Kodak Tri X film at exposure times of 90-120 seconds (Thoongsuwan and Cox, 1977a). Tests of IX, ABSA, and wholeglobulin containingboth IX and ABSA for passive induction of nonspecific acquired resistanceagainst RIA in rats and malaria in mice Four groups of 16 mature rats were each inoculated with 1 m1 of a 1:100 dilution of blood from a rat infected for 4 days with RIA agent (Thoongsuwan and Cox, 1977a). 0n following day one group of 16 rats was injected (IP) each with 1 ml of normal rat serum (NRS). A second group was given 1 ml of 198 globulin (9.0 mg of protein) from rats hyperimmunized by B, rodhaini infections. The third group each received 1 ml (9.0 mg protein) of the 7S fraction of globulin from the hyperimmunized rats. The fourth group was each given 1 m1 (26.5 mg protein) of whole globulin from hyperimmunized rats. A group of 8 uninfected rats was each given 1 ml of the whole hyperimmune globulin. Erythrocyte counts were made daily on the 8 rats from each of the infected groups as well as the uninfected control rats. Mortality in each group was recorded. 129 The same hyperimmune rat globulin and its pooled fractions were used to perform the experiment in mice. Four groups of 10 mice were each infected with 2 x 105 mouse erythrocytes parasitized with P. chabaudi as described (Cox and Milar, 1968). These and a group of 10 uninfected mice were given experimental injections on day l and day 3 of the experiment. The control mice were each injected IP with 0.5 ml of whole globulin (13.25 mg of protein) on each day. The first group of 10 infected mice were given 2 injections of 0.5 m1 of NRS. Two groups of 10 infected mice were given 0.5 ml injections (4.5 mg protein) of the 198 and 7S fraction of hyperimmune globulin respec- tively, and a fourth group of 10 infected mice were each given 0.5 m1 of the whole globulin from the hyperimmune rats. Blood for erythrocyte counts and staining for parasite quantitation was obtained daily by snipping the tail of the mouse with scissors. Erythrocyte and para- site counts were made microscopically as described (Thoongsuwan and Cox, 1973). Mortality was observed and recorded daily. EXPERIMENTAL RESULTS Studies of serum of rats with acute babesiosis and serum of recovered and hyperimmunized rats Figure 1 represents the results of Sephadex G-200 gel fil- tration profiles of globulin fractions from rats with acute B, rodhaini infection. IK activity was associated with the 195 peak showing a maximum titre of 5120. IR was not detected in the 78 or 45 fractions. CAH had been previously absorbed from the globulin prior to gel filtration and was not detected in the column fractions. ABSA 130 was not detected by immunodiffusion assays in any of the column fractions. The UV and 1K profiles of a sample of globulin from hyper- immunized rats are shown in Figure 2. 1K activity was again associ- ated with the 195 peak although the titres were lower than those associated with acute infection. ABSA was detected in fractions representing the 78 peak. ABSA was not detected in individual 19$ fractions or the concentrated 19S pool. Neither IK nor ABSA was detected in the 48 region (Figure 3). IX activity as detected with cellular intermediates was shown in Figure 4. The immunoconglutination titre of the globulin with EAC143 was 1280. No activity was detected with EAC14. Z-ME treat- ment of the globulin fraction destroyed IK activity. Tests on rats and mice passively immunized with 195 and 7S pools and with whole globulin of rats hyperimmunized by B. rodhaini infections for nonspecific acquired resistance The results of erythrocyte counts of uninfected rats injected with whole immune globulin, and of rats infected with RIA agent that were injected with 198 pool (IX), 78 pool (ABSA) and whole globulin (IX and ABSA), are shown in Table 1. Injection of whole immune globulin appeared not to affect the erythrocyte counts of uninfected rats. The infected rats given the 193 (IK) pool developed anaemia at a rate that did not differ from infected rats given normal rat serum, all rats in each group died of RIA within 8 days. Infected rats given the 7S (ABSA) pool and whole hyperimmune globulin (IX and ABSA) both had erythrocyte counts that were significantly lower on 131 days 3 and 4 than those of the infected rats given normal rat serum or 198 (IK) pool. On these same days the counts of the rats given whole globulin (IX and ABSA) were significantly lower than those given ABSA alone. Four of the 16 rats given ABSA alone and 7 of 16 given both IX and ABSA recovered from RIA. The erythrocyte counts of uninfected mice injected with whole immune rat globulin and mice infected with P, chabaudi and given injections of normal rat serum, IK, ABSA, or IX and ABSA are shown in Table 23. The mean percentage of parasitized erythrocytes (% PE) of the infected mice are shown in Table 2b. The injection of hyperimmune whole globulin appeared to have no effect on the erythrocyte counts of uninfected mice. The development of anaemia and parasitaemia in infected mice injected with NRS or with 198 (IK) did not differ and none of the mice in these groups were alive on day 9. Infected mice given whole globulin had significantly lower erythrocyte counts than those given NRS from day 2 through day 6, and their counts were lower than were those of mice given ABSA alone from day 2 through day 4. After day 6 the erythrocyte counts of mice that were to recover from P, chabaudi malaria or that would have prolonged survival, appeared to skew the mean of the erythrocyte counts. The % PE of mice given both IX and ABSA was lower than that for mice injected with NRS on day 4, and on day 5 through day 7, the % PE of the mice given ABSA alone was also lower than in the mice injected with NRS on day 5 through day 7. Longevity of 5 of 10 mice given ABSA was extended as was the longevity of mice given IX and ABSA. Three of the 10 mice in the latter group survived the malarial infection. 132 Immunofluorescent reactions of FITC conjugated IK and ABSA with blood films and spleen impression slides from rats with acute babesiosis, malaria, trypanosomiasis and RIA The reaction of ABSA-free conjugated IK with blood and spleen slides from rats made anaemic by each infection are shown in Figure 5. The reaction of the 7S (ABSA) pool is shown in Figure 6. Neither of the conjugates gave fluorescent activity with blood films or spleen impression slides from normal rats. Both preparations reacted with erythrocytes from the peripheral circulation and with those sequestered in the engorged spleens from the rats made anaemic by each infection. DISCUSSION In a companion communication, it was shown that rats that had recovered from infectious anaemia initiated by one agent had an acquired nonspecific resistance to infectious anaemia initiated by heterologous agents. The resistance, manifested by early anaemia, early reductions in parasitaemia, and by enhanced survival of the recovered rats after challenge, was accompanied by early elevations in the pre-existing titres of IX. However, elevations in the titres of CAH were also observed after challenge in each infection and it was suggested that the blood of the recovered rats may have contained ABSA (Thoongsuwan et al., 1977). We have studied the globulin (50% SAS fraction) of rats with acute babesiosis and of rats that had recovered and were then hyper- immunized by B, rodhaini infections. Finding all of the IK activity of globulin of rats with acute babesiosis and of the hyperimmunized 133 rats confined to the 19S fraction and was susceptible to inactivation by 2—ME treatment. This evidence suggests that IK activity generated in rats during and after infection with B, rodhaini was associated with IgM. This result is supported by others (Lachmann, 1967; Lachmann and Coombs, 1965). Another supportive result shown here is the specific reaction of IX against fixed C3 as demonstrated previously by Lachmann (1962) and Lachmann and Coombs (1965). However, the failure of rat IK under these experimental conditions to react with fixed C4 is not supported by the result obtained with rabbit IK as reported by Lachmann (1966). Since CAH had been removed from the globulins tested in these experiments, IK was distinctly identified with IgM and ABSA was found to be clearly associated with the 7S fraction, it was indicated that these antibodies are each distinct one from the other. In the passive transfer experiments in which IX and ABSA in the nonspecific acquired resistance associated with recovery from infectious anaemia, there was no evidence that IK alone made a con— tribution. However, infected animals passively immunized with 75 (ABSA) did exhibit the resistance, and among infected animals given injection of whole globulin from hyperimmunized rats, the nonspecific resistance appeared to have been enhanced. The resistance was mani- fested as reduced parasitaemia accompanied by early anaemia and by reduction or delay in mortality of the passively immunized animals. Since this nonspecific resistance was induced by injection of immune factors generated by B, rodhaini infection and was effective against infection with filterable RIA agent and with infection of P. chabaudi, 134 it is suggested that it was the result of humoral antibody lacking specificity for parasite antigen (5). The reaction of FITC conjugated 7S (ABSA) with circulating blood cells and with cells sequestered in the spleen from anaemic rats indicated that the antigen for ABSA (SA) had been elaborated in each infection. Finding that SA was elaborated in acute RIA and trypanosomal infections as well as in acute malaria and babesiosis tends to support the suggestion of Cox and Iturri (1976) that SA is self-antigen. These experiments also indicated that SA was bound to the surface of circulating erythrocytes as well as those sequestered in the spleen. Soni and Cox (1975b) found that after injection of plasma containing SA and ABSA into normal chickens SA could be detected on the surface of erythrocytes of the injected birds with FITC con- jugated ABSA. They postulated that soluble complexes of SA and ABSA became nonspecifically bound to erythrocytes and mimicked opsonin, causing these cells to be sequestered and phagocytized in the spleen and other filter organs as suggested by Dixon (1966). We interpret the reaction of ABSA observed in the present studies in the same way, i.e., the conjugate had reacted with the SA moiety of a SA-ABSA complex bound to the blood cells. Reactions of IK-FITC conjugate with blood and spleen slides from rats made anaemic by each of the infections were also noted. These results suggest that complexes of SA and ABSA were bound to the cells. These complexes have apparently fixed C3 as indicated by positive reactivity with fluorescent IK. It is possible that FITC conjugated IK may be useful for indicating the presence of 135 complement-fixed antigen-antibody complexes in vivo, but the reaction would have limited application in that it would tell one little about the offending antigen and antibody. The absence of anaemia in uninfected rats or mice injected with immune globulin containing both IX and ABSA suggested that these autoantibodies of themselves had no effect. This was to be expected since these animals did not contain the antigen that could react with ABSA and, in turn, fixed the complement to react with IK. Similarly, it is suggested that no effect on the infected animals injected with IR alone could be seen until both SA and ABSA had been elaborated for forming complexes. 0n the other hand, ABSA injected into infected animals was present when SA was elaborated, and it is suggested that complexes were formed earlier by several days than they were in animals injected with NRS or 1K alone. Since the action of complexes binding to cells is apparently nonspecific, it is suggested that infected as well as uninfected cells, or even free parasites in the blood, would react with complexes which would subsequently be sequestered and phagocytized, or to be lysed if optimal complement fixation was attained. This postulated mechanism might account for the early anaemia and reduced parasitaemia seen in the recovered rats after challenge (Thoongsuwan et al., 1977) which might account for the early anaemia and reduced parasitaemia seen in the infected animals injected with globulin containing ABSA. The enhancement of this activity in the animals given both IK and ABSA could be attributed to the conglutinating action of IX reacting with complement-fixed complexes that were bound to cells or parasites. 136 In considering this autoantibody-associated phenomenon of nonspecific acquired resistance of infectious anaemia, it should be remembered that soluble complexes of SA and its antibody were impli- cated as major pathogenic factors (Soni and Cox, 1974; 1975a; 1975b; 1975c). It is suggested that a significant part of the resistance presently reported might be a result of early removal of antigen by the antibody which kept the concentration of soluble complexes at a sublethal level. SUMMARY Immunoconglutinin (IK) from rats with acute babesiosis and from recovered-hyperimmunized rats was shown, by agglutination of complement-fixed sensitized sheep erythrocytes, to be 198 and 2- mercaptoethanol sensitive Ig which reacted specifically with fixed C3 fragment of complement. In addition to IK, blood of rats with acute babesiosis con- tained cold-active haemagglutinin (CAH) and soluble serum antigen (SA) and its antibody (ABSA). In hyperimmune rats, ABSA was associ- ated with 78 1g and CAH was not found. Thus CAH, IX and ABSA appeared to be distinct antibodies. Rats infected with filterable rat infectious anaemia (RIA) agent and mice infected with P, chabaudi were more resistant than controls after passive immunization with ABSA, but not with IK alone. The resistance was enhanced in animals given both ABSA and IX. The reactions of fluorescein conjugated ABSA with blood films and spleen impression slides from rats with acute RIA, babesiosis, 137 malaria and trypanosomiasis indicated that SA had been elaborated in each infection and was bound to blood cells. Reactions of conjugated IK with these slides suggested the presence of a cell bound complement- fixing immune complex. It is suggested that the cell bound complex mimicked Opsonin causing the sequestration of erythrocytes and parasites, and that this action was enhanced by immunoconglutination. This mechanism could account for the early anaemia and reduced parasitaemia in the nonspecific acquired resistance associated with infectious anaemias. REFERENCES Chan, P. E. G., and H. F. Deutsch. 1960. J. Immunol. 85:37-45. Cox, H. W. 1957. J. Immunol. 79:450-454. Cox, H. W., and G. Calef. Iturri. 1976. Ann. Trop. Med. Parasitol. 70:73-79. Cox, H. W., and R. Milar. 1968. Amer. J. Trop. Med. Hyg. 17:173-179. Dias da Silva, W., and I. H. Lepow. 1967. J. Exp. Med. 125:921-946. Dixon, F. J. 1966. Mil. Med. (Suppl.) 131:1233-1234. Goldman, M. 1968. Fluorescent antibody methods. Academic Press, New York. Lachmann, P. J. 1962. Immunol. 5:687-705. Lachmann, P. J. 1966. Immunol. 11:263-271. Lachmann, P. J. 1967. Adv. Immunol. 6:479-527. Lachmann, P. J., and R. R. A. Coombs. 1965. In: Complement (Ed. by G. E. W. Wolstenholme and J. Knight). Little, Brown 8 Co., Boston, pp. 242-273. Linscott, W. D. 1975. J. Immunol. 105:1625-1630. 138 Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. J. Biol. Chem. 193:265-275. Musoke, A. J., H. W. Cox, and J. F. Williams. 1977. Am. J. Trop. Med. Hyg. 26:(in press). Nilsson, U. R., and H. J. Muller-Eberhard. 1965. J. Exp. Med. 122:277-298. Rapp, H. J., and T. Borsos. 1963. J. Immunol. 91:826-832. Soni, J. L., and H. W. Cox. 1974. Amer. J. Trop. Med. Hyg. 23: 577-585. Soni, J. L., and H. W. Cox. 1975a. Amer. J. Trop. Med. Hyg. 24: 206-213. Soni, J. L., and H. W. Cox. 1975b. Amer. J. Trop. Med. Hyg. 24: 423-430. Soni, J. L., and H. W. Cox. 1975c. Amer. J. Trop. Med. Hyg. 24: 431-438. Thoongsuwan, S., and H. W. Cox. 1973. Ann. Trop. Med. Parasitol. 67:373-385. Thoongsuwan, S., and H. W. Cox. 1977a. Ann. Trop. Med. Parasitol. (submitted). Thoongsuwan, S., and H. W. Cox. 1977b. Ann. Trop. Med. Parasitol. (submitted). Thoongsuwan, S., H. W. Cox, and R. A. Patrick. 1977. Trans. Roy. Soc. Trop. Med. Hyg. (submitted). 139 o~.o H ~m.~ v~.o H ow.~ mm.o H mu.m m mm.o H 0H.m mH.o H Ho.N mH.o H co.» m ow.o H oH.n om.o H vm.N m~.o H mo.m mH.o H mn.~ om.o H mm.» m wm.o H on.v mv.o H mm.v um.o H mo.v o~.o H Hw.m om.o H on.w o mm.o H 5H.n Hm.o H mv.n n~.o H 05.5 mm.o H Ho.“ NH.o H Nv.w m mHN.o H mm.n mH.o H Hm.n 0H.o H we.» NN.o H ne.w m~.o H Hm.m v n.mw~.o H mH.n H~.o H mu.“ em.o H mv.w o~.o H om.w wH.o H Hv.w m n.mv~.o H no.“ Hm.o H ~m.w -.o H nn.w mH.o H wH.w v~.o H ov.w N H~.o H co.» mm.o H Hm.w mH.o H me.» wH.o H vn.w vH.o H o~.w H NN.o H av.m o~.o H wm.w wH.o H mm.w m~.c H mm.» mv.o H mm.n o cHHsnoHu :oHHuomcH oz ...mwo ..m~ .mmH .mmz cHasnoHu :3 oHomg mxmo :HHz wouooncH was vouoomcH Houuaoo .m.m H ooH x 0mm .msoum some :H mama 0H macaw mnuaov co vommn mm: mumu vopuomcH mo museum :H NHHHHHHoz .maoum some :H mumn w :o owns one: mussou ouxoonnuxuo :moz .cHHsnon oasssHHomx: oHonz :HHz was cHHnnon ocsaaHHomxg mo :oHuomum Ho .mmoH Ho mo.o v a pm unmoHMHcmHmn .msonm mmz Ho>o .mmoH Ho mo.o v a an acmonchHma. .vo>H>H:m :o>om .oH xwv :o usto .n xmv :o voHv Han o=o«a« .vo>H>H=m usom .oH xmu co oaH: .m xmv so voHv mama ooHAHHH .w xmv co voHv HHo11mmoH Ho mo.o v a HH unmoHMHcmHmp .moouw mzz Ho>o11mmoH Ho mo.o v m an HamonHcmHma .vo>H>Hsm oops» .NH xmv :o o>Hmn.o Han co voHv ooHa o3eHH¢ .NH xav no muovcHaaoH on» .m xau :o 03» .m Haw :o voHv ooHs oouzhac .m xxv co mnovcHHEQH on» .w xmv :o voHu macaw some :H ooHa oH mo ozhH 1 HH oe.o H mm.H NH :oHHoomcH oz ...HmwmnoHo ..ma .mmH .mmz eHHsnoHo oHo;z mxmo on: neocoan Haw HouoomeH Houueoo .m.m H ma H .HoscHHcoo11.m~ oHoHe illililllfu 145 Fig. l.--The optical density (0.D.) at 280 nm and the titres of immunoconglutinin (IK) in fraction samples after column chromatography with Sephadex G-200 of pooled, cold agglutinin- free, globulin from rats with acute Babesia rodhaini infection. 146 Sephadex G-200 Acute Rat Globulin §.rodholnl (CAH-Free) 0 02.0 nm ------ 1 K TITER TUBE NUMBER Figure 1 83M]. )1 l 147 Fig. 2.--The optical density (0.D.) at 280 nm, the titres of immunoconglutinin (IX) and the results of tests for antibody to serum antigen (ABSA) in fraction samples after column chromatography with Sephadex G-200 of pooled globulin from rats hyperimmunized by Babesia rodhaini infections. 148 2» Hana .. 111.11.] M. H a. .M .3 H: 1% H1... mmwm.» H wm_w m H \\\\\\\\\\ 111111111 1m11111 w flu ....................... HT “T. M... .1. E303 280 210 Fro TUBEIHMEER Figure 2 149 Fig. 3.--Photograph of immunodiffusion in gel test of 4S, 198, and 7S fraction pools obtained by Sephadex G-200 column chranatography of globulin from rats hyperimmunized by Babesia rodhaini infections, whole globulin of the hyperimmunized rats (HIBr), whole globulin from rats hyperimmunized by Plasmodium chabaudi infections (HIPC) and normal rat globulin (N) with serum ffom rats with acute B, rodhaini infection (Br). 150 151 Fig. 4.--Reactions of immunoconglutinin (IK), from 198 fraction pools globulin from rats with acute Babesia rodhaini infection which had been absorbed free of cold-agglutifiin, with sheep erythro- cytes (E), sensitized sheep RBC (EA), sensitized sheep RBC fixed with C14 (EAC14) and C143 (EAC143) fragments of human complement. ZME-IK = Z-mercaptoethanol treated 1K. Diluent control = No IK. 152 IK Titre o o g N g g B 2 (N ( ' o - (H ‘ . '3 IK 1‘, (I93) ':.\ x“ 0.": 2."; " , _ 0,09,19‘9< —2ME IK 1-0-9‘9 - - Cells— EAC|4 (9 9 9 9 9;9 9’09 9'991(9‘) Diluent EAC,43(o>.’6,’o o o o 0., 01.. one?) Control Figure 4 153 Fig. S.--Photomicrographs of immunofluorescent reactions of fluorescein isothiocyanate conjugate of CAH—free IK globulin from rats immunized with autologous serum-absorbed kaolin. A. With blood films from a normal rat (N), a rat with acute babesiosis (Br), a rat with acute malaria (PC), a rat with acute trypanosomiasis (T1), and a rat with acute rat infectious anaemia (V). B. With spleen impression slides from a normal rat (N), a rat with acute babesiosis (Br), a rat with acute malaria (Pc), a rat with acute trypanosomiasis (T1), and a rat with acute rat infectious anaemia (V). 154 FA: Kaolin- Serum Induced IK N A Blood Smears Figure 5 155 Fig. 6.--Photomicrographs of immunofluorescent reactions of fluorescein isothiocyanate conjugated 7S (ABSA) fraction of globulin from rats hyperimmunized by Babesia rodhaini infections. A. With blood films from a normal rat (N), a rat with acute babesiosis (Br), a rat with acute malaria (Pc), a rat with acute trypanosomiasis (T1), and a rat with acute rat infectious anaemia (V). 8. With spleen impression slides from a normal rat (N), a rat with acute babesiosis (Br), a rat with acute malaria (Pc), a rat with acute trypanosomiasis (T1), and a rat with acute rat infectious anaemia (V). 156 FA: 73 Hyperimmune Rat B.rodhoini Impressions Figure 6