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Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.

Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.

Resistance of adult C57BL/6 mice to severe Cryptosporidium parvum infection is dependent on CD4+alpha beta+ TCR lymphocytes. In this study, we demonstrated that treatment with anti-IFN-gamma mAb extended oocyst excretion 18 days longer, and anti-IL-4 mAb extended oocyst excretion at least 11 days longer than isotype control mAb treatment. Analysis of the specific activity of anti-IFN-gamma mAb present in treated mouse sera suggested that IFN-gamma may have a limited role in the resolution phase of infection. Changes were also documented in numbers of CD4+alpha beta+IFN-gamma+ and CD4+alpha beta+IL-4+ lymphocytes in Peyer's patches and intraepithelium of adult C57BL/6 mice during resolution of C. parvum infection. Resistance to initial severe infection was associated with CD4+alpha beta+IFN-gamma+ lymphocytes, and eventual resolution of infection was associated with CD4+alpha beta+IL-4+ lymphocytes. Analysis of cytokine expression following in vitro stimulation with C. parvum Ags during resolution of infection demonstrated consistent increases in CD4+alpha beta+IL-4+ lymphocytes, but not CD4+alpha beta+IFN-gamma+ lymphocytes. The relevance of CD4+alpha beta+IL-4+ lymphocytes in protection against C. parvum was then evaluated in C57BL/6 IL-4 gene knockout mice (IL-4(-/-)). Adult IL-4(-/-) mice excreted oocysts in feces approximately 23 days longer than IL-4(+/+) mice. Further, anti-IFN-gamma mAb treatment increased the severity and the duration of infection in IL-4(-/-) mice compared with those in IL-4(+/+) mice. Together, the data demonstrated that IFN-gamma was important in the control of severity of infection, and either IFN-gamma or IL-4 accelerated termination of infection. However, neither IL-4 nor IFN-gamma was required for the final clearance of infection from the intestinal tract of adult mice.

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.

Development of the rickettsia, Anaplasma marginale, in salivary glands of male Dermacentor andersoni exposed as nymphs or adult ticks, was studied indirectly by inoculation of susceptible calves with homogenates and directly by examination, using light microscopy and a DNA probe; some unfed ticks were incubated before tissues were collected. Salivary gland homogenates made from ticks in every treatment group caused anaplasmosis when injected into susceptible calves; prepatent periods decreased as the time that ticks had fed increased. Colonies of A marginale were seen only in salivary glands of ticks exposed as adults and not in those exposed as nymphs; the percentage of salivary gland acini infected in these ticks increased linearly with feeding time. However, the probe detected A marginale DNA in salivary glands of ticks from both groups; the amount of DNA detected increased as feeding time was extended. The amount of A marginale DNA appeared to remain constant in gut tissues, but to increase in salivary glands. Salivary glands of adult-infected male ticks that were incubated, but did not feed a second time, became infected with A marginale, and the pattern of infection of acini varied with incubation temperature. Development of A marginale in salivary glands appears to be coordinated with the tick feeding cycle; highest infection rate was observed in ticks exposed as adults.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.

The envelope (env) genes of two antigenic variants of caprine arthritis-encephalitis virus (CAEV), defined by serum neutralization, were expressed in vaccinia virus. Recombinant gp135 envelope glycoprotein competitively inhibited neutralizing activity of serum from CAEV-infected goats, indicating gp135 is a dominant target antigen of CAEV neutralizing antibody. In addition, type-specific neutralizing activity of goat serum directed against one variant was inhibited by both homologous and heterologous variant recombinant gp135. Hence, a CAEV variant env gene encodes type-specific neutralization epitopes of both variants. The results indicate that antigenic variation of CAEV involves env gene mutations encoding amino acid differences outside conserved neutralization epitopes affecting epitope exposure to the immune system.

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.

Virus replication and subsequent viremia are clearly correlated with clinical disease in EIAV infected horses. Termination of viremia is the result of specific immune responses. Recurrences of viremia are associated with antigenic variation of neutralization-sensitive epitopes. Immunosuppression experiments indicate that the eventual control of EIAV and development of carriers is mediated by the immune system. Even though the immune response to EIAV has a protective effect, immune responses also cause some of the lesions. At least one part of the anemia, erythrocyte destruction, is caused by the immune response. Not all of the mechanisms of decreased erythropoiesis are known, but EIAV infection of monocyte/macrophages results in altered iron metabolism and functional iron deficiency. Viral antigen-antibody-C3 complexes cause glomerulitis and the combination of antigen-antibody reactions results in significant reductions in plasma C3. Lesions in the liver and other organs are infiltrations of lymphocytes and monocytes/macrophages in the interstitial areas and it is assumed that these lesions are initiated by specific immune responses to viral antigens. The observations on kinetics of EIAV infection, immune control by the horse, and immunopathologic basis of most of the lesions lead to the conclusion that mechanisms of lentivirus control and disease can be determined by study of EIAV.

Rabbits were immunized with purified caprine arthritis-encephalitis virus and examined for neutralizing activity. Analysis of virus-antiserum interaction at 37 degrees C demonstrated little loss of viral infectivity after incubation with heat-inactivated rabbit antiserum for 60 min. However, sensitization of virus (as assessed by the addition of complement) occurred almost immediately and was 95% complete after 10 min. The complement-dependent neutralizing activity was associated with the immunoglobulin G fraction of rabbit antiserum. Addition of goat anti-rabbit immunoglobulin G to the immune rabbit serum-caprine arthritis-encephalitis virus mixture also resulted in neutralization of infectivity when unbound antibody was removed before addition of the anti-immunoglobulin. Serum from most caprine arthritis-encephalitis virus-infected goats contains antibody activity to the core protein p28, as demonstrated by immunodiffusion and enzyme-linked immunosorbent assay. However, attempts to demonstrate neutralizing activity in the serum of goats up to 1.5 years post-inoculation or in serum of hyperimmunized goats were unsuccessful when the sera were examined alone or in combination with complement or rabbit anti-goat immunoglobulin or both.

Variable surface antigens of cloned populations of African trypanosomes were studied using 2-dimensional gel electrophoresis and monoclonal antibodies. Two-dimensional gel maps showed that the only major differences in protein profiles of the non-nuclear materials from different clones of solubilized trypanosomes were attributable to the variable surface antigens and that purification doesn't alter these molecules, at least with respect to charge and apparent m.w. Purified variable surface antigens were used as immunogens and a number of monoclonal antibodies were derived using cell-fusion technology. In radioimmunoassay, each of the monoclonal reagents was shown to be specific for the immunizing antigen, and in immunofluorescence tests using acetone-fixed trypanosomes, each bound specifically to the clone from which the antigens were purified. Only 20% of the monoclonal reagents bound to living trypanosomes, however, providing evidence for both exposed and nonexposed antigenic sites on the variable surface antigens. Those reagents derived to a single antigen bound to different nonoverlapping antigenic sites, which were on the protein portion of the molecules and not the carbohydrate moieties. The data show that monoclonal antibodies are ideal probes for studying localization of antigenic sites on the antigen molecules and for studying antigen synthesis, glycosylation, and architecture in relation to the cell surface. In addition, the finding of exposed and nonexposed antigenic sites on trypanosome variable antigens allows a possible explanation for the role of the antigens in pathogenesis of African trypanosomiasis.

The first demonstration of immune serum-mediated neutralization of Anaplasma marginale and identification of the involved surface antigens is presented. Anaplasma marginale initial bodies were purified from parasitized erythrocytes by using ultrasonic disruption and differential centrifugation. The initial bodies were morphologically intact, as determined by electron microscopy, were not agglutinated by anti-bovine erythrocyte sera, and were infective. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated minimal erythrocytic contamination in the purified initial bodies. Rabbit antisera against initial bodies completely neutralized the infectivity of purified initial bodies for splenectomized calves, whereas normal rabbit serum did not alter infectivity. Nine initial body proteins were surface radioiodinated by using lactoperoxidase, and five of these were precipitated by the neutralizing antibody (105,000, 86,000, 61,000, 36,000, and 31,000 apparent m.w.). The results demonstrate that initial body determinants capable of inducing neutralizing antibody are present. The identification of surface radiolabeled proteins immunoprecipitated by the neutralizing antibody provides candidates to be tested as protective immunogens in cattle.

The transfer of circulating immunoglobulin G1 (IgG1) antibody to the gastrointestinal tract in young calves was quantified by using bovine anti-dinitrophenol IgG1 antibody labeled with 125I. The antibody was administered to newborn calves by intravenous injection, and transfer of the labeled IgG1 to the gastrointestinal tract occurred as demonstrated by excretion of protein-bound label in the feces and by the presence of the labeled IgG1 antibody in the gastrointestinal tract lumen at necropsy. Sixty-eight percent of the [125I]IgG1 clearance occurred by transfer to the gastrointestinal tract. Protein-bound 125I in the gastrointestinal tract lumen retained 65% of the specific dinitrophenol-binding ability of the labeled antibody originally administered. These results show that (i) transfer to the intestinal lumen is the major means of IgG1 clearance in calves, and (ii) this transfer results in antigen-binding antibody in the intestinal tract lumen. The potential contribution to enteric immunity of IgG1 reaching the intestinal lumen from circulation remains to be determined.

Babesia bigemina is one of several tick-borne hemoparasitic diseases of cattle that are inadequately controlled and cause substantial livestock production losses in tropical and subtropical climates. Recovery from acute babesiosis is associated with development of protective immunity against subsequent challenge with both homologous and heterologous parasites. Viable and infectious merozoites, the intraerythrocytic stage of B. bigemina responsible for clinical disease, were separated from contaminating host cells by density gradient centrifugation. Monoclonal antibodies developed against gradient-separated merozoites were screened for surface reactivity against live merozoites in an immunofluorescent binding assay. Surface-reactive antibodies immunoprecipitated five major biosynthetically radiolabeled merozoite proteins with relative m.w. of 72,000, 58,000, 55,000, 45,000, and 36,000 in SDS-PAGE. Two additional proteins immunoprecipitated with the 45,000 m.w. protein were unreactive with monoclonal antibody in western blots and are apparently part of a membrane complex co-precipitated by this antibody. In contrast, additional proteins of m.w. of 36,000, 35,000, and 33,000, immunoprecipitated with the 58,000 protein, all contain the surface-exposed epitope bound by monoclonal antibody. Immune serum from an animal that had recovered from infection with a Mexico isolate of B. bigemina immunoprecipitated five radiolabeled proteins from the Mexico isolate that co-migrated in SDS-PAGE with major proteins precipitated by surface-reactive monoclonal antibodies. In addition, antibodies against a Kenya isolate of B. bigemina immunoprecipitated the same co-migrating proteins from radio-labeled Mexico isolate, demonstrating epitope conservation between surface proteins of geographically different isolates. The identification of proteins with epitopes exposed on the surface of live merozoites and accessible to antibody provides candidates to be tested as protective immunogens in cattle.

Cryptosporidiosis is a diarrheal disease of humans, calves, and other mammals caused by the coccidian parasite Cryptosporidium parvum. Immune bovine serum and two surface-reactive antisporozoite mAb with neutralizing activity were used to identify sporozoite surface Ag by radioimmunoprecipitation/SDS-PAGE and immunoblotting. When isolated sporozoites were incubated with mAb 18.44, 12 to 25 times the ID50 for mice was completely neutralized. This mAb binds diffusely to the sporozoite surface and recognizes a sporozoite surface Ag that eluted in the void volume of a Bio Gel A column with an exclusion limit of 500,000 daltons. The Ag recognized by mAb 18.44 was not radiolabeled with 125I or [35S] methionine, migrated with the dye front in SDS-PAGE, and was insensitive to proteinase K digestion, suggesting a non-protein composition. mAb 17.41 significantly neutralized 25 times the ID50 of sporozoites for mice. This mAb binds multifocally to the sporozoite surface and recognizes [35S] methionine-labeled sporozoite surface Ag of 28,000 m.w., 55,000 m.w., and 98,000 m.w. Immune bovine serum immunoprecipitated [35S] methionine- or 125I-labeled sporozoite Ag ranging from less than 14,300 m.w. to greater than 200,000 m.w., including surface Ag of 28,000 m.w. and 55,000 m.w. The results indicate that two different molecules capable of inducing neutralizing antibody are exposed on the surface of C. parvum sporozoites.

The effect of circulating passive antibody on immunity to bovine rotavirus infections in neonatal calves was investigated. In the first experiment, rotavirus antibody titers in the small intestinal lumina of 5- and 10-day-old calves with a wide range of serum rotavirus antibody titers were determined. Neutralizing antibody was present in the small intestinal lumina in titers that correlated with the calves' serum titers (r = +0.84, P less than 0.01). Immunoglobulin G1 was the predominant isotype of intestinal luminal rotavirus antibody. Calves not fed colostrum during the absorptive period lacked rotavirus antibody in circulation and in the intestinal lumen at 7 days of age, even when they were fed large volumes of colostrum with a high rotavirus antibody titer at 48 h after birth. Therefore, rotavirus antibody is not retained in the intestinal lumen for 5 days following a colostrum meal, and the luminal antibody in the 5- and 10-day-old seropositive calves were probably derived from circulating antibody. In a second experiment, calves were passively immunized by subcutaneous injection of colostral whey with a high immunoglobulin G1 rotavirus antibody titer and challenged with virulent bovine rotavirus 48 h later. The passively immunized calves were protected from rotavirus infection and diarrhea compared with calves with comparable serum immunoglobulin concentrations but with lower serum rotavirus with lower serum rotavirus antibody titers. The results of these experiments indicate that circulating immunoglobulin G1 antibody appears in the gastrointestinal tract of neonatal calves and that circulating rotavirus antibody can prevent infection and diarrhea after rotavirus challenge.

A 14- to 26-kilodalton fraction of Staphylococcus aureus exoproteins isolated by molecular sieve chromatography and electroelution from polyacrylamide gels was shown to specifically react with antibodies in milk of naturally infected dairy cows. Silver staining of the antigen preparation electrophoresed in polyacrylamide gels showed the strongest reactivity in the 24- to 26-kilodalton region with lesser staining at lower apparent molecular sizes. An enzyme-linked immunosorbent assay was developed to differentiate infected from uninfected cows for diagnostic purposes. Samples from S. aureus-infected cows reacted in the assay, and samples from uninfected cows did not. There was no correlation between numbers of somatic cells in the samples and reactivity to the antigens. Samples from cows infected with coagulase-negative staphylococci did not react with the antigens. It was found, however, that some samples from uninfected cows that were recently postpartum or producing low amounts of milk contained antibodies which bound the antigens. This was believed to be due to transport from blood to the mammary gland of antibodies which were generated by previous intramammary infections or infections at other sites.

Disease is a major constraint in small ruminant production systems in lesser-developed countries throughout the world. Animal health projects have been an integral part of the Small Ruminant Collaborative Research Support Program (SR-CRSP) from its inception. At the onset, these projects were oriented toward herd health care and veterinary extension activities. Later, all the projects developed a sharper focus in that they were directed to more basic studies of infectious disease. Diseases currently being investigated include caseous lymphadenitis, contagious caprine pleuropneumonia, caprine arthritis-encephalitis, ovine pulmonary carcinoma, ovine progressive pneumonia and neonatal mortality of alpaca. Continued, sharply focused studies are projected for the future to take advantage of recombinant technology in the development of multivalent vaccines.

The pathogenesis of the anemia in 17 horses experimentally infected with the virus of equine infectious anemia (EIA) was investigated. It was demonstrated by direct Coombs' tests that 15 of the animals had C′3 but no IgG, IgM or IgG(T) on their RBC surfaces. Red blood cells from 8 of 10 horses with a positive anti-C′3 Coombs' test were more osmotically fragile than normal. The RBC life spans of 7 horses with C′3 coated RBC ranged from 18 to 90 days (normal 136 ± 17 days). It was concluded that the hemolysis in this virus disease was associated with C′3 coating of RBC.

The role of non-complement-fixing anti-equine infectious anemia (EIA) antibody in the conversion of complement fixation (CF) tests from positive to negative in EIA-infected horses was investigated. Complement-fixation inhibition (CFI) tests demonstrated antibodies in sera that were CF negative. These antibodies would bind to antigen, but would not fix complement. The inhibiting antibodies were isolated and shown to be IgG(T) by immunoelectrophoresis and immunodiffusion against monospecific anti-IgG(T) antisera. Separation of immunoglobulins from affected horse sera by DEAE cellulose chromatography revealed 11 of 13 had complement-fixing IgG and non-complement-fixing IgG(T) antibodies present simultaneously regardless of the reactivity of whole sera. The reactivity of whole sera in CF and CFI tests evidently depended upon the relative concentration and perhaps affinity of the Ig classes. The predominant class fluctuated in serial sera from four of six persistently infected horses.

There is increasing evidence that complement-dependent immune reactions are pathogenetically important in virus diseases. One indication of in vivo complementfixing immune reactions is the lowering of plasma C3 levels. A study was made of the circulating plasma C3 levels in 12 horses infected with equine infectious anemia (EIA) virus. Purified equine C3 was prepared from euglobulin precipitates of horse serum using triethylaminoethyl (TEAE) column chromatography with subsequent Pevikon block electrophoresis. Monospecific antisera prepared in rabbits was used in single radial immunodiffusion to quantitate C3 levels. A significant reduction of C3 was detected in 6 of 12 horses with active clinical disease. All horses with reduced C3 levels had C3-coated erythrocytes and deposits of immunoglobulin and C3 in the glomeruli. These findings further indicate that immune processes are active in the pathogenesis of EIA.

The results of a study on the induction of IFN-alpha, IFN-beta, and IFN-gamma in normal and SCID foals showed a deficiency of IFN-gamma but not IFN-beta in SCID foals. The ability of SCID mononuclear cells to produce IFN-alpha in response to poly I:C but not to NDV may indicate a partial deficiency of IFN-alpha in SCID foals. The deficiency of IFN-gamma and presence of IFN-beta in SCID foals supports the classification of IFN-gamma and IFN-beta as immune and nonimmune interferons, respectively. Furthermore, the deficiency of IFN-gamma in SCID foals may in part explain the high susceptibility to infections observed in SCID foals.

Foals with combined immunodeficiency had normal levels of purine salvage pathway enzymes, including adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase.

Concentrations of immunoglobulin (Ig) G1 were measured in 590 sera from 24- to 36-hour-old lambs, and failure in passive transfer (FPT), less than 6 mg/ml, occurred in 20 lambs. Of the 20 FPT lambs, 45% died before 3 weeks of age, whereas only 5% of the 570 lambs with adequate passive transfer died before 3 weeks of age. The low percentage of FPT was attributed to management practices ensuring suckling by the lambs and possibly to influences from several years of selecting ewes on the basis of weaned lamb production. The correlation between the concentration of IgG1 in 257 postpartum, presuckle ewe colostrum samples and the IgG1 concentrations in 362 lamb sera from those ewes was low (r = 0.32). However, the mean serum IgG1 concentration in 20 lambs from ewes with the lowest postpartum, presuckle colostrum IgG1 concentrations (less than 30 mg/ml) was significantly lower (P less than 0.001) than mean serum IgG1 concentration in 24 lambs from ewes with the highest postpartum, presuckle colostrum IgG1 concentrations (greater than 110 mg/ml). Postpartum, presuckle colostrum IgG1 was measured in 7 ewes whose lambs had FPT, and the IgG1 values varied throughout the colostrum IgG1 range. Colostrum IgG1 concentrations could not be used to explain FPT or to identify ewes likely to have lambs with FPT.

Studies were conducted to determine the mechanisms of antibody suppression of phytohemagglutinin (PHA)- and concanavalin A (Con A)-induced lymphocyte proliferation. Initial experiments incubated lymphocytes with either PHA or Con A, and at various times the respective anti-phytolectin was added to the cultures. DNA synthesis was less than 20% of the total response if anti-PHA was added within 2 hr and if anti-Con A was added within 10 hr of addition of mitogens to the culture. At the concentrations used, anti-PHA reduced PHA binding to the lymphocytes, while anti-Con A did not reduce Con A binding to the cells. Antibody was non-toxic and specific for its respective mitogen. Additional experiments incubated lymphocytes with either PHA or Con A and then the cells were washed and placed in mitogen-free media. The addition of antibody to cultures which had been washed markedly suppressed proliferation. Using radiolabeled PHA and Con A in one group of experiments and fluorescent labeled anti-mitogen antibodies in others, it was determined that antibody did not remove Con A or PHA from the cell, but instead antibody slowed the release of mitogen from the lymphocyte. Anti-mitogen antibody remained attached to mitogen on the surface of lymphocytes for at least 24 hr. The experiments suggest that antibody can suppress the lymphocyte reaction by blocking the necessary phytolectinlymphocyte interaction and interrupting stimulation by cell-bound mitogen.

Variable surface antigens of cloned populations of African trypanosomes were studied using 2-dimensional gel electrophoresis and monoclonal antibodies. Two-dimensional gel maps showed that the only major differences in protein profiles of the non-nuclear materials from different clones of solubilized trypanosomes were attributable to the variable surface antigens and that purification doesn't alter these molecules, at least with respect to charge and apparent m.w. Purified variable surface antigens were used as immunogens and a number of monoclonal antibodies were derived using cell-fusion technology. In radioimmunoassay, each of the monoclonal reagents was shown to be specific for the immunizing antigen, and in immunofluorescence tests using acetone-fixed trypanosomes, each bound specifically to the clone from which the antigens were purified. Only 20% of the monoclonal reagents bound to living trypanosomes, however, providing evidence for both exposed and nonexposed antigenic sites on the variable surface antigens. Those reagents derived to a single antigen bound to different nonoverlapping antigenic sites, which were on the protein portion of the molecules and not the carbohydrate moieties. The data show that monoclonal antibodies are ideal probes for studying localization of antigenic sites on the antigen molecules and for studying antigen synthesis, glycosylation, and architecture in relation to the cell surface. In addition, the finding of exposed and nonexposed antigenic sites on trypanosome variable antigens allows a possible explanation for the role of the antigens in pathogenesis of African trypanosomiasis.

Twelve chemicals and commercial disinfectants were tested for inactivation of equine infectious anemia virus. In the presence of 10% bovine serum, all chemicals inactivated 4 log10 (based on 0.1 ml) of the virus within 5 minutes at 23 C. A reduction of at least 4 log10 was observed when the virus was exposed for 1 minute to substituted phenolic disinfectants (3 commercial preparations and sodium orthophenylphenate), halogen derivatives (iodophor and sodium hypochlorite), chlorhexidine, and 70% ethanol. Sodium hydroxide (5%), 2% formalin, and 2% glutaraldehyde were slower to inactivate the virus, but achieved 4 log10 reduction in titer by 5 minutes' contact time. The susceptibility of the equine infectious anemia virus to chemical disinfectants is similar to that of other enveloped viruses.

Serum IgG1 concentrations of calves less than 3 weeks old and dying from infectious disease were significantly lower (P less than 0.01) than those of clinically normal calves. Fifty percent of the dead calves had serum IgG1 concentrations that were more than 2 standard deviations below the normal mean, and an additional 35% had IgG1 concentrations that were more than 1 standard deviation below the normal mean. Low IgG1 concentrations were attributed to failures in passive transfer of colostral immunoglobulin. The few calves dying of noninfectious causes generally had normal serum immunoglobulin concentrations. The results of this study emphasize the importance of adequate colostral intake and absorption to the neonatal calf. In view of the large numbers of calves that die from neonatal infection each year, it may be assumed that failure in passive transfer, as reflected by low serum immunoglobulin concentrations, is one of the most important factors influencing neonatal calf mortality.

Goats vaccinated with inactivated caprine arthritis-encephalitis virus (CAEV) developed more severe arthritis after infectious CAEV challenge exposure than did goats vaccinated with tissue culture medium. Arthritis also developed more rapidly in the group vaccinated with inactivated virus. In another experiment, goats with persistent CAEV infection developed acute arthritis after at least 2 injections of infectious CAEV at monthly intervals. In this experiment, the control group consisted of goats with persistent CAEV that were given tissue culture medium. Seemingly, the immune response to CAEV is an important cause of the CAEV-induced arthritis.

Presuckle colostral samples and lamb serum samples taken 36 h postpartum were assayed for immunoglobulin G1 (IgG1) concentration (mg/ml) using single radial immunodiffusion. Breeds sampled included Polypay (P), Rambouillet (R), Targhee (T), Columbia (C), Finnish Landrace (F) and Finn crosses (Fx). Sources of variation examined in IgG1 concentration in colostrum (dam trait) included dam's sire breed, dam's sire, age of ewe and number of lambs born. All sources of variation were statistically significant. Least-squares means of IgG1 levels for sire breed were 80, 64, 67, 64, 72 and 69 mg/ml for P, R, T, C, F and Fx breed groups, respectively. A fetal stimulus may exist to increase the mass of IgG1 in colostrum available for multiple births (61, 69 and 77 mg/ml for single, twin and triplet, respectively). Ewe age was a significant source of variation because of a high mean concentration of IgG1 in the yearling's colostrum (100 mg/ml), whereas only slight differences occurred between the other age groups (65 to 67 mg/ml), except for the 7-yr older group (53 mg/ml). Sources of variation examined in IgG1 concentration of lamb serum at 36 h postpartum (lamb trait) included lamb's sire breed, lamb's sire, age of dam, birth type and sex, with dam's colostral IgG1 concentration and day born as covariates. Sire within breed, birth type and the two covariates were significant. Least squares means for sire breed were 36, 32, 33, 32, 31 and 32 mg/ml of serum for P, R, T, C, F and Fx groups, respectively. Lamb serum IgG1 decreased as birth type increased. The heritability of IgG1, estimated by paternal half-sib analyses, was .19 +/- .12 for colostrum and .18 +/- .06 for lamb serum.

Combined immunodeficiency (CID), a defect in both B- and T-lymphocytes, was found to occur in 2.3% of 257 foals of Arabian breeding. All affected foals died by 5 months of age. The belief that CID is transmitted as an autosomal recessive genetic defect was supported by results from matings of dams and sires that had previously produced affected foals. Based on a prevalence of 2.3%, the proportion of carriers of the CID trait among the adult population surveyed was estimated to be 25.7%. Recent descriptions of other immunologic defects in foals emphasized the need for careful differential diagnosis. Disorders that could be confused with CID include failure in passive transfer of immunoglobulins from dam to foal, via colostrum; transient hypogammaglobulinemia; agammaglobulinemia (associated with B-lymphocyte deficiency and normal T-lymphocytes), and selective IgM deficiency.

Summary Summary. In-vitro culture of Haemophilus somnus in liquid or solid media supplemented with bovine blood or serum resulted in non-immune binding of immunoglobulin (Ig) by the organism. This binding was independent of the antigen-combining site of the Ig molecule, since binding of an IgG preparation specific for the hapten dinitrophenol was unaffected by the presence of the homologous antigen. Quantitative comparison of the binding of Ig fragments Fab and Fc demonstrated that the non-immune binding occurred in the Fc region of bovine IgG. The isotypes of Ig that became bound to H. somnus included both bovine IgG subclasses (IgG1 and IgG2), which were bound equally, and bovine IgM.

The results from several experiments demonstrated that an effective vaccine for contagious caprine pleuropneumonia could be made with inactivated F38 mycoplasma. Evaluation of the amounts of lyophilised F38 mycoplasma plus saponin showed that the optimum formulation was 0.15 mg of mycoplasma in saponin. Saponin inactivates the mycoplasma and provides the adjuvant effect necessary to stimulate a protective immune response. The lyophilised F38 mycoplasma could be stored for 14 months at either 4 degrees C or 22 degrees C without losing its immunising potential. A single immunisation with the optimum formulation produced a protective immune response in goats that lasted for longer than one year.

125I-labeled immunoglobulin (Ig)G1 in colostral whey was used to determine the region of maximum absorption of Ig from the small intestine of the neonatal calf and the variation in Ig absorption among calves at the intestinal level. In experiment 1, 5 segments (approx 5%, 35%, 60%, 80%, and 95% of the duodenocecal length) were formed in the small intestine of 9 colostrum-deprived calves shortly after birth. These segments were injected with colostral whey containing 125I-IgG1 4 hours after birth, and uptake, transfer, and absorption (defined as uptake plus transfer) were determined for each segment 2 hours later. Raw data were adjusted for the milligrams of IgG1 injected per gram of intestinal tissue to obtain the least squares mean (LSM) value. The LSM values for absorption of IgG1 from distal segments 3, 4, and 5 were significantly greater (P less than 0.05) than those values for proximal segments 1 and 2. The region of the maximum IgG1 absorption was the lower small intestine, 60% to 80% of the duodenocecal length. There was also an indication of independence between uptake and transfer in each of the segments. Significant differences (P less than 0.05) were present among calves in the LSM values for uptake and absorption, but not for transfer. In experiment 2, thoracic ducts of 8 newborn calves were cannulated 4 to 5 hours after birth. At 6 hours after birth, colostral whey with 125I-IgG1 was injected into an intestinal segment (approx 60% to 80% of the duodenocecal length).(ABSTRACT TRUNCATED AT 250 WORDS)

The consequences of experimental infection with Trypanosoma congolense and later superinfection with T brucei were compared among four breeds of goats: Toggenburg, Nubian, Alpine and Saanen. No major differences were found with regard to packed cell volume, bodyweight or parasitaemia. When these four breeds were considered for introduction into areas having African trypanosomiasis, none appeared to have a comparative advantage with regard to innate resistance to trypanosomes.

Latex beads were sensitised with a polysaccharide isolated from a F38 culture supernatant and used in a slide agglutination test to detect serum antibodies in goats with contagious caprine pleuropneumonia. The latex agglutination test detected antibodies in the sera of goats by 22 +/- 2 (mean +/- 1 sd) days after contact exposure to contagious caprine pleuropneumonia, whereas the complement-fixation test detected antibodies by 24 +/- 4 days after contact exposure. Both tests were negative with 181 sera from a farm which was free of the disease. When the same tests were done on 763 sera from two different farms with outbreaks of classical contagious caprine pleuropneumonia, 63 per cent were positive by the latex agglutination test and 23 per cent were positive by the complement-fixation test. Besides being more sensitive than complement fixation, the latex agglutination test can be performed in the field using undiluted serum or whole blood and a result obtained within two minutes.

Bovine immunoglobulin G subclass (IgG1 and IgG2) antibodies were found to fix bovine complement while only IgG1 fixed guinea-pig complement in vitro. Similar results were noted when IgG1 and IgG2 antibodies were tested by passive cutaneous anaphylaxis (PCA) in that both IgG1 and IgG2 caused PCA in bovine skin while only IgG1 mediated the reaction in rat skin. In precipitation reactions IgG1 antibodies to DNP failed to cause precipitation of DNP19-ovalbumin while IgG2 antibodies to DNP precipitated DNP19-ovalbumin. Both IgG1 and IgG2 antibodies to ovalbumin precipitated ovalbumin. Surprisingly, IgG2 antibodies to equine erythrocytes caused phagocytosis by bovine neutrophils and peripheral blood monocytes while IgG1 antibodies failed to cause either phagocytosis or adherence. Results with peripheral blood monocytes cultured for 7 days demonstrated that both IgG1 and IgG2 could mediate phagocytosis.

Crias, newborn alpacas (Lama pacos), that were almost agammaglobulinemic at birth had a 70% increase in total serum proteins within 24 hours largely because of absorption of gamma globulins from colostrum. Immunoglobulin G was the isotype in highest concentration in colostrum and in serum from 24-hour-old crias. The serum IgG concentration of 10 crias increased linearly (r = 0.97) from a mean of 0.3 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 30.1 mg/ml (+/- 8.1 SD) at 24 hours. The 24-hour concentration decreased by half in 10 days. Immunoglobulin M also was absorbed from colostrum and increased linearly (r = 0.99) from a mean of 0.5 mg/ml (+/- 0.1 SD) for serum collected before crias suckled to a maximal mean of 4.2 mg/ml (+/- 2.2 SD) 24 hours after birth. The 24-hour serum concentration of IgM decreased by half in 7 days. Therefore, on a weight basis, 7 times more IgG than IgM was transferred to crias; IgG accounted for greater than 85% of the passively transferred proteins in serum of 24-hour-old crias. Absorption of functional antibodies of IgG and IgM isotypes from colostrum of immunized dams by crias also was demonstrated. Immunoglobulin G and IgM antibody titers to chicken RBC increased linearly to maximal geometric mean titers of 1,139 and 843, respectively, 24 hours after birth. The 24-hour IgG and IgM antibody titers decreased by half in 6 and 3.8 days, respectively. Purified alpaca IgG had a molecular mass of 166 kilodaltons, a predominant gamma mobility, and an extinction coefficient of 14.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Partially purified thymus products were used to evaluate the maturation of T lymphocytes in immunodeficient mice. Three different bovine thymic extracts, designated thymic extracts A, B, and C, were successful in increasing the longevity of conventionally raised nude mice. Daily injection of bovine thymic extracts A, B, and C and mouse thymus extracts failed to mature a population of T lymphocytes and restore the capacity to reject heart allografts. Preincubation of normal syngeneic bone marrow with thymic extract B in vitro before injection into nude mice also failed to reconstitute the host's ability to reject heart grafts. The number of antibody plaque-forming cells of sheep red blood cells in lethally irradiated, bone marrow-reconstituted mice could be increased by preincubating the bone marrow cells with bovine thymic extract fraction B before injection followed by daily injections. A similar but less marked increase in plaque-forming cells was obtained by the daily injection of bovine thymic extract fraction C. Complete functional maturation of T lymphocytes in immunodeficient animals appears to require more than thymic extract stimulation of bone marrow cells or pre-T lymphocytes.

Babesia bigemina is one of several tick-borne hemoparasitic diseases of cattle that are inadequately controlled and cause substantial livestock production losses in tropical and subtropical climates. Recovery from acute babesiosis is associated with development of protective immunity against subsequent challenge with both homologous and heterologous parasites. Viable and infectious merozoites, the intraerythrocytic stage of B. bigemina responsible for clinical disease, were separated from contaminating host cells by density gradient centrifugation. Monoclonal antibodies developed against gradient-separated merozoites were screened for surface reactivity against live merozoites in an immunofluorescent binding assay. Surface-reactive antibodies immunoprecipitated five major biosynthetically radiolabeled merozoite proteins with relative m.w. of 72,000, 58,000, 55,000, 45,000, and 36,000 in SDS-PAGE. Two additional proteins immunoprecipitated with the 45,000 m.w. protein were unreactive with monoclonal antibody in western blots and are apparently part of a membrane complex co-precipitated by this antibody. In contrast, additional proteins of m.w. of 36,000, 35,000, and 33,000, immunoprecipitated with the 58,000 protein, all contain the surface-exposed epitope bound by monoclonal antibody. Immune serum from an animal that had recovered from infection with a Mexico isolate of B. bigemina immunoprecipitated five radiolabeled proteins from the Mexico isolate that co-migrated in SDS-PAGE with major proteins precipitated by surface-reactive monoclonal antibodies. In addition, antibodies against a Kenya isolate of B. bigemina immunoprecipitated the same co-migrating proteins from radio-labeled Mexico isolate, demonstrating epitope conservation between surface proteins of geographically different isolates. The identification of proteins with epitopes exposed on the surface of live merozoites and accessible to antibody provides candidates to be tested as protective immunogens in cattle.

The capacity of cells from thymus, liver, spleen, mesenteric lymph nodes, peripheral blood, and bone marrow to respond to in vitro phytolectin and allogeneic lymphocyte-stimulation was determined in 16 pony fetuses 61 to 200 days old (gestational age). Phytolectin-responsive cells were detected in the thymus at the 80th gestational day, peripheral blood at 120 days, lymph node at 160 days, and spleen at 200 days. Mixed lymphocyte culture-responsive cells were detected in thymus at 100 days and in the spleen at 200 days (gestational age). Immunoglobulins (Ig) M and IgG were quantitated by radioimmunoassay. They were detected in fetuses prior to 200 days of age. All of 50 normal newborn foals had detectable quantities of IgM (165 +/- 56 micrograms/ml of serum). Quantities of IgG in normal newborn foal serum were lower and more variable. The minimal-maximal concentrations of IgG were 2 to 170 micrograms/ml of serum with a mean and SD of 51 +/- 49 micrograms/ml. The results indicate that (1) functional T lymphocytes are present in the fetus by 100 days (gestational age), (2) functional B lymphocytes are present by 200 days, and (3) that foals are immunocompetent before birth.