Lymphocyte blastogenesis and cellular cytotoxicity in a congenital infection of bovine fetuses related to epizootic bovine abortion

The disease referred to as epizootic bovine abortion (EBA) was experimentally induced in bovine fetuses. Dark-field microscopy was used to detect congenital infection with an unclassified spirochaete-like organism. Some of the fetuses collected at abattoirs were also found to be naturally infected with a morphologically similar microorganism. Blood counts and organ weights were correlated with the presence of the microorganism. Lymphocyte blastogenesis increased, the result of in vivo stimulation among the infected fetuses. Phytomitogens (phytohaemagglutinin, concanavalin A and lipopolysaccharide) also stimulated greater responses in infected fetuses when compared to results in normal fetuses. Cellular cytotoxicity was examined by the single cell assay and results indicated that there were fewer cytotoxic lymphocytes among the diseased fetuses. The infected abattoir-collected specimens were obtained from clinically normal adult cattle, and the immunological changes in these fetuses were closely characterised with those of the EBA diseased fetuses. These naturally infected fetuses showed signs of a mild infectious disease.     

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.     

Histopathologic changes in bovine fetuses after repeated reintroduction of a spirochete-like agent into pregnant heifers: association with epizootic bovine abortion

A spirochete-like organism was found in the plasma of bovine fetuses affected with epizootic bovine abortion (EBA). The spirochete-like organism was frequently found in abattoir-collected fetuses as an inapparent infection, and EBA was found in cattle on foothill rangeland where the vector tick Ornithodorus coriaceus could repeatedly reintroduce the infectious agent into pregnant cattle (superinfection). Epizootic bovine abortion resembled a naturally acquired superinfection in circumstances where the agent was frequently present in the environment under conditions favoring transmission. Therefore, to determine whether fetal lesions could be experimentally induced in utero, spirochete-like organisms collected from clinically normal fetuses at an abattoir were inoculated IV and subcutaneously into 2 pregnant heifers 5 times over a 4-month period to mimic repeated tick transmission in the field. Macroscopic and microscopic examinations of tissues from 2 cesarean-collected fetuses and from 3 calves born at term with the naturally acquired spirochete infection indicated that the calves had evidence of an infection that caused morphologic changes compatible with immunologic stimulation and mild reticuloendothelial hyperplasia. Compared with findings in the calves, lesions in the superinfected fetuses were more severe, and the lesion distribution in various organs was more extensive. The spirochete-like organism appeared to be a mild pathogen because of its persistence in the host. Clinical disease from the infection may only develop with repeated superinfections. Therefore, a relationship between this microorganism and EBA is probable.     

Involvement of calcium in mitogenic and immunoglobulin responses in bovine lymphocytes

Removal of calcium from cultures of bovine splenic lymphoid cells with ethyleneglycol-bis-(2-amino ethylether)-N,N'-tetraacetic acid (EGTA) caused significant suppression of pokeweed mitogen-induced immunoglobulin (Ig) biosynthesis. It was found that removal of calcium for 48 hours of a 5-day culture significantly altered Ig biosynthesis. Re-addition of calcium after 48 hours did not restore Ig synthesis. The EGTA caused significant (P < 0.05) suppression of DNA synthesis when calcium was removed from concanavalin A- and purified protein derivative-stimulated peripheral blood lymphocyte cultures. Further, removal of calcium for 48 hours of 4-day cultures significantly altered the DNA synthesis in mitogen- and antigen-stimulated cultures.     

Protective effects against abortion and fetal infection following exposure to bovine viral diarrhea virus and bovine herpesvirus 1 during pregnancy in beef heifers that received two doses of a multivalent modified-live virus vaccine prior to breeding

Objective-To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection. Design-Randomized controlled clinical trial. Animals-33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves. Procedures-20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days. Results-After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers. Conclusions and Clinical Relevance-Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.     

Diagnostic survey of bovine abortion with special reference to Neospora caninum infection: importance, repeated abortion and concurrent infection in aborted fetuses in Southern Brazil

The protozoa Neospora caninum is an important cause of bovine abortion world-wide. The objective of this survey was to determine the distribution pattern of infectious abortion in Southern Brazil with special reference to N. caninum infection. A total of 161 bovine aborted fetuses from 149 farms were analysed during a 1.5 year period. The cause of abortion was identified in 51.5% of cases. Overall, 23% (37/161) of the fetuses were considered to be infected with N. caninum. Bacterial infection accounted for 17.4% (28/161) of cases, fungal infection for 3.1% (5/161) of cases and viral aetiology for 1.8% (3/161). Six fetuses had concurrent infection with N. caninum and Leptospira spp. Data from 111 fetuses and the respective aborted cows were analysed to investigate the association between previous abortion and current N. caninum infection. The prevalence of N. caninum-infected fetuses from cows with and without a history of previous abortion was 44% (11/25) and 24.4% (21/86), respectively. Cows aborting a N. caninum-infected fetuses were 2.4 times more likely to have aborted previously than cows aborting for other reasons (95% CI of odds ratio=0.9-6.8, P=0.06).     

Surface markers for characterisation of bovine blood lymphocyte populations and changes in these from birth to maturity

A sheep erythrocyte (E) rosette technique was developed for use with cattle lymphocytes. This involved the use of 17 per cent Ficoll 400 and preservative-free heparin (84 iu/ml) in the saline-erythrocyte mixture. Using this technique, 83 per cent of peripheral blood lymphocytes in cattle aged between six and 10 years were found to form E rosettes. The remaining cells (17 per cent) were B-cells, so that no cells remained unmarked. Lymphocytes from very young calves contained a population of unmarked or null cells, but these rapidly diminished as the animals matured. A peak of total lymphocytes recovered from blood, as well as E rosette-forming cells, occurred in calves aged four to six months. The non-E rosette-forming cells were mostly B-cells and it was suggested that this was associated with calf weaning. The total number of lymphocytes recovered, as well as E rosette-forming cells, gradually fell with the age of the cattle sampled. Null cells were virtually absent from the blood of cattle six years and older. Bovine T-cells could be further subdivided into Fc mu, Fc gamma and C' receptor-bearing subpopulations on the basis of overlap with R rosette-forming cells. Some further separation of these cells from B-cells was achieved using density gradient centrifugation on Percoll. Separation of E rosette-forming cells with Fc receptors from E rosette-forming cells without Fc receptors was achieved by nylon wool columns, to which the Fc receptor bearing cells were adherent. It was concluded that bovine blood lymphocytes had blood T-lymphocyte populations with markers which may correspond to the 'helper' (Fc mu ) and 'suppressor' (Fc gamma ) populations described for the human.     

Fetal and placental lesions in bovine abortion due to Tritrichomonas foetus

Laboratory records and histologic material from 13 cases of naturally occurring, early- to late-term bovine abortion due to Tritrichomonas foetus were examined. Placentitis was present in all placentas. Pyogranulomatous bronchopneumonia was in six of the 11 lungs which were examined. Trichomonad organisms were demonstrated within the placental stroma in all cases and free or phagocytized in pulmonary airways in seven cases.     

Ontogeny of immunity and leukocytes in the ovine fetus and elevation of immunoglobulins related to congenital infection

Certain humoral and cellular aspects of the immune response were observed during gestation in the ovine fetus. Emergence and development of leukocytes in peripheral blood were observed. Morphologically mature lymphocytes were consistently present at 32 days of gestation, but mature neutrophils were not neutrophils were not consistently present until gestational day 123. Monocytes were first observed at 63 days and eosinophils at 112 days of gestational age. Numbers of each class of leukocyte increased from the time of their appearance until just prior to birth when they declined. Production of immunoglobulin G (IgG) and immunoglobulin M (IgM) was monitored from the time of their appearance at 56 and 77 days, respectively, until birth. In contrast to consistently low base-line values of IgG and IgM (below 0.22 mg/ml and 0.21 mg/ml, respectively) found in clinically normal, unstimulated fetuses, the immunoglobulin values in experimentally infected animals increased with the age of the fetus. This finding can be utilized in diagnosis of several congenital infections and anomalies.     

Fatal congenital anaplasmosis associated with bovine viral diarrhoea virus (BVDV) infection in a crossbred calf

Clinical disease resulting from the vertical transmission of Anaplasma marginale has only been reported on 5 occasions despite studies demonstrating successful in utero transmission. During the reported experimental induction of congenital anaplasmosis in calves, the outcome was variable but mostly led to inapparent or mild infection. There are previous case reports of fatal congenital anaplasmosis following natural infection. The clinical findings in a 2-day-old calf presented to the Onderstepoort Veterinary Academic Hospital with clinical signs of congenital anaplasmosis, which was unresponsive to treatment, are described. Subsequent post mortem diagnostic tests revealed that this calf was co-infected with bovine viral diarrhoea virus (BVDV). It is postulated that immunosuppression resulting from BVDV infection predisposed to severe, fatal anaplasmosis in this calf.