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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.

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.

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.

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.

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)

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.

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.

Failure of passive transfer (FPT) of immunoglobulin from colostrum was demonstrated as a major determinant of mortality in newborn alpacas (Lama pacos; crias). Serum IgG concentrations of dying crias were significantly (P less than 0.0001) lower than were serum IgG concentrations of crias that lived. Of 82 crias, 10 (12%) died within 1 month of age, and 7 of these had 0 to 9 mg of IgG/ml of serum at 48 hours after birth; 5 of the 7 had evidence of infectious diseases. The serum IgG concentrations of the remaining dead crias were 12, 13, and 20 mg/ml. On the basis of serum IgG concentrations of crias that died in the first month, FPT was defined as a 48-hour serum IgG concentration less than 9 mg/ml, which was greater than 2 SD below the 48-hour mean of clinically normal crias. Using this definition, the prevalence of FPT in the 82 crias studied was 9%. Corroborative evidence of the relationship between FPT and mortality was obtained from a retrospective study of 21 dead crias. The postmortem serum IgG concentration of 5 crias that died 2 to 10 days after birth ranged from less than 1 to 3 mg/ml; all were greater than 2 SD below the mean of age matched clinically normal crias. The range of serum IgG concentration was 2.2 to 21 mg/ml in 8 crias that died 11 to 20 days after birth; serum IgG concentration in 1 cria was greater than 2 SD below the normal mean, and 6 were greater than 1 SD below the normal mean.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated equine immunoglobulin (Ig)G(T) antibodies to equine infectious anemia virus P26 antigen did not precipitate with antigen when the ratio of antibody to antigen was high. However, at lower ratios of antibody to antigen precipitation occurred. In addition, complement-fixation by IgG and P26 antigen was inhibited by high concentrations of IgG(T). The unusual reaction pattern noted with IgG(T) antibodies was still detectable by the immunodiffusion test for equine infectious anemia virus. In situations of nonprecipitability by IgG(T), the adjacent positive control line was inhibited, and this was interpreted as a positive reaction.

Studies were conducted to determine the ways in which antibody regulates proliferation of lymphocytes stimulated by phytohemagglutinin (PHA) and concanavalin A (ConA). Equine lymphocytes were reacted with PHA or ConA for 1 to 10 hours, washed free of excess lectin, then cultured for 3 days and examined for proliferation and incorporation of [3H]thymidine. Both PHA and ConA induced lymphocytes to proliferate after short incubation periods. This response could be interrupted by the addition of antibody to the respective lectin. Total suppression of the proliferative response occurred if antibody was added within the first 2 hours after initial contact with PHA or ConA. Addition at times up to 48 hours produced either total or partial suppression. The removal of excess antibody 4 hours after addition to the culture did not reverse the suppression. Using radiolabeled ConA and PHA, it was observed that suppressive antibody inhibited the release of lectin from metabolically active lymphocytes and from surfaces where pinocytosis was not possible or was minimized. Antibody did not affect the rate of degradation of lectin by the cells. These results indicate that antibody attached to lectins, already bound to the lymphocyte surface, can terminate the lymphocyte response and retard the rate of release of lectin from the cell membrane.

Sixty-six cases of combined immunodeficiency (CID) in foals were studied to determine the most prevalent causes of infection and death. Lesions of the respiratory system were observed in 59 of the foals and were attributable to infection with equine adenovirus. Pneumocystis carinii, and bacteria. Significant lesions were also observed in liver, pancreas, intestines, heart, and kidneys. Maintenance of foals with CID for experimental purposes is directed at the prevention and control of these secondary infections. Adenovirus can be controlled by administration of horse plasma containing high titers of antiadenovirus antibody. Bacteria are controlled by appropriate antibiotic therapy. Pneumocystis carinii infection remains a significant problem in the maintenance of foals with CID.

Caprine arthritis-encephalitis is a retrovirus-induced disease resulting in lymphoproliferative lesions of the CNS and joints. Peripheral blood leukocytes of chronically infected goats were analyzed for the types of cells present and for their reactivity to viral antigen and polyclonal stimulants. Two of 9 infected goats had abnormal numbers of B lymphocytes--one elevated and the other deficient. Lymphocyte reactivity to viral antigens was transiently detectable by a lymphoblastogenic assay in 5 of the 9 goats. The reactive cells were peanut agglutinin-negative T lymphocytes. Concanavalin A induced more division in T lymphocytes of infected goats than in lymphocytes of noninfected goats, whereas the reactions to phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide were no different in the 2 goat groups. It is concluded that goats infected by the caprine arthritis-encephalitis virus have antigen-reactive T lymphocytes and that infection promotes the response to a nonspecific T-cell stimulant.

Delayed hypersensitivity (DH) responses of normal and immunodeficient horses were evaluated with antigens [dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH)] and phytolectins [phytohemagglutinin (PHA), concanavalin A (Con A)]. Immunologically normal horses sensitized with 5 daily applications of 2 mg of DNCB developed positive skin reactions upon challenge with 0.4 mg of DNCB. The delayed onset of the reaction and the predominately mononuclear cell infiltration at the test site indicated these were DH reactions. Normal horses sensitized with 500 microgram of KLH and challenged with 100 microgram of KLH developed skin reactions appearing earlier and composed of neutrophils and mononuclear cells, suggesting involvement of both humor and cellular mechanisms. Arabian foals with combined immunodeficiency failed to respond visibly to either antigen. Intradermal injection of 50 microgram of PHA induced visible reactions in all normal horses tested. The slow development and the predominately mononuclear cell infiltrate of PHA reactions resembled DH responses to antigen. Reactions were induced by Con A in 15 of 16 normal adult horses and 7 of 14 normal foals. The reactions were rapid in onset and contained numerous eosinophils. All 6 foals with combined immunodeficiency failed to respond to PHA injection, whereas 5 of 6 did not respond to Con A. Based on the results of these studies, it appears that in vivo skin tests with PHA and DNCB can be used to screen horses for deficient T-lymphocyte responses.

The in vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes (PBL) from ponies were studied. Prednisolone inhibited lymphocyte stimulation by phytohemagglutin (PHA) in a dose-dependent manner, without inducing lysis even at large doses. The PBL from horses heterozygous for the combined immunodeficiency trait responded to corticosteroid treatment the same as did PBL from normal ponies. Removal of the corticosteroid after incubation with PBL from normal ponies partially restored responsiveness of these cells to PHA. Chronic in vivo treatment of ponies with corticosteroids caused a marked decrease in the absolute numbers of circulating lymphocytes. Most remaining lymphocytes had detectable surface immunoglobulin and C3 receptors, suggesting a greater decrease in the T-lymphocyte population. In spite of this, there was little change in the in vitro PHA- or keyhole limpet hemocyanin-sensitized ponies. In general, the corticosteroid effects of lysis, as well as the mitogenic and antigenic responses of PBL from ponies, were similar to those previously reported for human lymphocytes.

Caprine arthritis-encephalitis virus was isolated from goat milk and transmitted most efficiently to kids through both milk and colostrum. In addition, it appeared that transmissions through other secretions of the doe occurred, but were less important than transmission in milk and colostrum. Intrauterine infection may have occurred in 2 of 32 cesarean-derived goats, but postpartum horizontal transmission could not be ruled out. Transmission by the aerosol route was not demonstrated, and even short-term direct contact between virus-infected bucks and virus-free does during breeding did not result in transmission. Prolonged direct contact for over 12 months between weaned cesarean-derived goats and virus-infected goats was necessary before horizontal transmission could be demonstrated under nondairy conditions. However, when uninfected does were milked with infected does, a high percentage became infected in less than 10 months. Heat inactivation (56 C) reduced approximately 10(5) median tissue culture infective doses of caprine arthritis-encephalitis virus to below titratable levels, and virus was not transmitted to kids fed virus-infected colostrum that had been heated at 56 C for 1 hour. A program of eradication is discussed in which kids are removed from their dams at birth, fed safe sources of colostrum and milk, and isolated from other goats until weaning.

The lentiviruses, caprine arthritis-encephalitis virus (CAEV) and progressive pneumonia virus (PPV) of sheep, cause major diseases in their respective hosts; however, the infectivity of these viruses for closely related species has not been determined. Experiments were conducted to determine whether CAEV would infect sheep and whether PPV would infect goats. Upon inoculation with CAEV, lambs developed a nonsuppurative arthritis and antibody to CAEV, and the virus was isolated up to 4 months later. Exposure of 3 lambs to CAEV-infected adult goats did not lead to demonstrable infection after 18 months. Young goats inoculated with PPV replicated the virus and developed arthritis and antiviral antibody. These results demonstrate that these distinctly different lentiviruses may infect and cause diseases in species other than their accustomed host. Presently used techniques may not be effective in differentiating which lentivirus is responsible for infection of sheep and goats. Our results also indicate that mixing sheep and goats may adversely influence attempts to eradicate lentiviruses from these species.

Nine monoclonal antibodies which reacted with equine immunoglobulin (Ig)M and not other equine Ig and serum proteins were prepared. Cells producing antibodies (C 1.9) which precipitated with IgM and bound to staphylococcal protein A were triple-cloned (C 1.9/3.2) and the antibodies further characterized. Monoclonal antibody C 1.9/3.2 reacted with an IgM determinant present on serum IgM from horses of several breeds. Studies with 125I-labeled IgM revealed the presence of this determinant on all IgM molecules. The monoclonal antibody enabled quantitation of IgM in presuckling foal and adult horse sera, using rocket electrophoresis. This procedure was used because presumably it gives a positive precipitation reaction over a wide range of antigen-antibody ratios. The C 1.9/3.2 monoclonal antibody recognized an exposed mu-chain determinant on live B lymphocytes, as determined by immunofluorescence. Also, IgM-containing cells could be identified in acetone-fixed frozen sections of lymphoid tissue. Sera from several other species carry the determinant identified by C 1.9/3.2, suggesting that the reagent may be useful for IgM studies in other species.

Laboratory methods designed to quantitate serum immunoglobulin were evaluated: single radial immunodiffusion, zinc sulfate turbidity, and serum electrophoresis. Estimation of immunoglobulin concentration in neonatle calves, using total protein measurements, was also evaluated. Single radial immunodiffusion proved useful for quantitation when either class or subclass information was needed. Zinc sulfate turbidity measurements gave accurate results for total immunoglobulin except when hemolysis was present in the sample. A correction factor for hemoglobin that will minimize errors resulting from hemolysis was presented. Serum electrophoresis was also found to be an accurate quantitation method. Large errors were encountered in attempting to estimate immunoglobulin on the basis of total protein.

Agammaglobulinemia was diagnosed in a 1-year-old Thoroughbred horse on the basis of the following observations: (1) absence of serum immunoglobulins M, A, and G(T); (2) small amounts of serum immunoglobulin G (16 mg/100 ml); (3) absence of specific antibody in the serum of the horse following immunization and challenge exposure to 2 antigens; (4) absence of plasma cells, primary follicles, and germinal centers in a lymph node removed after antigenic stimulation; (5) absence of "natural" serum antibodies to rabbit-erythrocytes which were easily detectable in age-matched control horse serums; and (6) increased susceptibility to infections. There was evidence of functional cell-mediated immunity which included a skin response to injected phytolectins, skin response to antigen challenge following sensitization, and in vitro proliferative response of lymph node cells to phytohemagglutinin. An intact cell-mediated immune response was also supported by the observation that the horse lived to 17 months of age without antibody production, whereas horses with an absence of both antibody production and cell-mediated immunity (combined immunodeficiency) die by 4 months of age without immunologic intervention. The known features of agammaglobulinemia in this horse are similar to those in sex-linked agammaglobulinemia in persons and are unique among the immunodeficiences described in other animals.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-gel immunodiffusion.