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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.

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.

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.

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.

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.

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.

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)

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.

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.

A sodium sulfite-precipitation test utilizing 3 concentrations of sodium sulfite to evaluate the immune status of neonatal calves allowed scoring of serum immunoglobulin concentrations as less than 5 mg/ml, 5 to 15 mg/ml, and greater than 15 mg/ml. Of the 54 estimations for serums that contained greater than 5 mg/ml, 96% were correct; of the 30 estimations for serums that had between 5 and 15 mg/ml, 90% were correct; and of the 42 estimations for for serums with greater than 15 mg/ml, 93% were correct. It was concluded that this test can be used under field conditions to evaluate the immune status of neonatal calves.

Arabian horses with severe combined immunodeficiency disease (SCID) were evaluated for thymic hormone activities using thymic extracts and sera. Extracts prepared from thymus of SCID horses were able to increase the number of spleen cells responding to sheep red blood cells in irradiated, bone marrow-reconstituted mice. In addition, ultrafiltrates prepared from sera of these immunodeficient horses, which contained material with molecular weight of less than 50,000 Daltons could (a) induce a population of human bone marrow precursor cells to differentiate into cells bearing SRBC receptors and form spontaneous E-rosettes, a characteristic of T lymphocytes, and (b) stimulate cyclic adenosine monophosphate (AMP) synthesis in mouse thymocytes. Based on in vivo and in vitro effects, it was concluded that the defect of these Arabian horses with severe combined immunodeficiency disease did not involve a complete thymic hormone inadequacy.

Forty-eight-hour serum concentrations of immunoglobulin G1 (IgG1) were determined for 123 female calves born on 1 farm during a 12-month period and fed 2.8 L of colostrum by esophageal feeder within 2 hours of birth. Mean monthly serum IgG1 concentrations were lowest in the winter, and increased during the spring and early summer to reach their peak in September, after which they decreased. Eight of 64 calves (12.5%) born from June through October had failure of passive transer of IgG1 (serum IgG1 less than 8 mg/ml), whereas 27 of 58 (46.5%) born from November through May had failure of passive transfer.

With regard to our studies on the functional properties of bovine IgG1 and IgG2 antibodies, the differences were minimal when homologous systems were evaluated. Both IgG1 and IgG2 fixed bovine complement by the classical pathway, caused homologous passive cutaneous anaphylaxis, caused phagocytosis of coated erythrocytes by cultured monocytes and precipitated with an antigen having multiple unique determinants (ovalbumin). In contrast to IgG2, IgG1 caused neither adherence nor phagocytosis by freshly isolated neutrophils and monocytes. Additionally, IgG2 antibodies to DNP precipitated with DNP19ovalbumin while IgG1 antibodies formed soluble complexes with this antigen.

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.

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.