Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies

Pregnant goats were inoculated intravenously or in uterine arteries with Brucella abortus, and tissues from the uterus and placenta were examined by electron microscopy. Identification of B. abortus in placentae was with antibody-coated colloidal gold. B. abortus was first seen in phagosomes of erythrophagocytic trophoblasts and in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Subsequently, trophoblast necrosis and ulceration of chorioallantoic membranes were present. Coincidently, B. abortus was present in the lumen of placental capillaries. In late stages of infection, placental vasculitis was present, and placentomal trophoblasts were separated from maternal syncytial epithelium. In lesions with vasculitis, large numbers of B. abortus were in connective tissue of chorionic villi. Within the placentome, trophoblasts that lined chorionic villi contained no intracellular bacteria and were separated from B. abortus by intact basement membranes. These results suggest that bacteremic B. abortus is endocytosed by erythrophagocytic trophoblasts and that B. abortus replicates in the rough endoplasmic reticulum of chorioallantoic trophoblasts. Replication of brucellae in trophoblastic rough endoplasmic reticulum is unique; we believe that B. abortus may utilize endoplasmic reticulum for synthesis and glycosylation of bacterial membrane proteins or that B. abortus catabolizes trophoblast secretory proteins.     

Pathogenesis of Brucella abortus infection of the mammary gland and supramammary lymph node of the goat

Goats, both in late pregnancy and soon after parturition, were inoculated intravenously with Brucella abortus, and mammary glands and supramammary lymph nodes were examined by light and electron microscopy at 2 to 55 days post-inoculation. After 7 days, lymphoplasmacytic, histiocytic interstitial mastitis with a lobular and periductal distribution were detected microscopically. Brucellae, identified in tissues with immunoperoxidase staining and antibody-coated colloidal gold stain, were first seen in macrophages and neutrophils throughout mammary parenchyma, but most commonly in mammary alveoli. In subsequent samples, infected phagocytes progressively increased in number, especially in ductal and alveolar lumina, and adjacent parenchyma. B. abortus was in phagosomes and phagolysosomes in macrophages and neutrophils; degenerate and necrotic phagocytes were often filled with brucellae. Extracellular brucellae were associated with ruptured necrotic infected phagocytes. Supramammary lymph nodes draining infected mammary glands were enlarged. Lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis were seen microscopically. Brucellae were seen exclusively in macrophages, which were most often located in subcapsular and cortical sinuses. This study suggests that phagocytic leukocytes 1) transport brucellae into mammary glands; 2) provide a site for intracellular replication in mammary secretions; and 3) transport brucellae from mammary glands to supramammary lymph nodes.     

Pathogenesis of Brucella abortus in chicken embryos

Chicken embryos inoculated with Brucella abortus at 6, 10, and 12 days of incubation were examined by light and electron microscopy. B. abortus was identified by avidin-biotin immunoperoxidase and immunogold techniques. Death occurred from 2 to 5 days post-inoculation, depending on age of the embryo and route of inoculation. B. abortus was recovered from all infected eggs. Brucellae had spread throughout all tissues and localized preferentially within cells of mesodermal derivation. Organ distribution and degree of bacterial replication varied with age of the embryo at time of inoculation. In 6-day-old embryos, B. abortus localized preferentially in endoderm and mesoderm of yolk sac wall, extra- and intraembryonic serosal epithelia, and glomeruli of the mesonephros. In 10- and 12-day-old embryos, B. abortus spread to all tissues; renal glomeruli, liver, spleen, and heart were most severely infected. Intracellular B. abortus was within the rough endoplasmic reticulum of mesenchymal, mesothelial, yolk endodermal, and hepatic cells. In mononuclear phagocytes, endothelial cells, and granulocytes, bacteria were within membrane-bound vacuoles. Intracellular replication of B. abortus in embryonic tissues, especially yolk endoderm, closely resembled that in experimental infections of trophoblasts.     

Lung lesions in bovine fetuses aborted by Brucella abortus.

Considering the poor facilities available for microbiological diagnosis in some countries where Brucella abortus is a frequent cause of bovine abortion, a study was conducted to determine if isolation of B. abortus from an aborted bovine fetus could be predicted from a detailed histological study of the formalized lung. Thirty-nine samples of B. abortus positive and 20 negative fetal samples were examined for the presence of 14 different pulmonary lesions. Differences in the frequency of observed lesions between the positive and negative groups, were determined by odds ratios and chi square statistic. The confidence of the prediction was calculated by means of the logistic computer model. The frequency of eight lung lesions was found to be significantly (p less than 0.05) different between the groups; nevertheless, these lesions were not specific enough to be able to incriminate B. abortus as the cause of abortion.     

Experimental infection of goat fetuses in utero with a stable, rough mutant of Brucella abortus.

Fetuses of goats in their last trimester of pregnancy were experimentally infected with Brucella abortus strain RB51, a stable rough mutant deficient in the perosamine O-chain content of its lipopolysaccharide. RB51 maintained its rough phenotype in vivo and did not induce abortion. Infection with RB51 resulted in the production of significant levels of IgG type antibodies specific for B abortus cellular antigens distinct from the perosamine O-chain. These findings suggest that strain RB51 will be useful in the pregnant goat for studying the role of brucella antigens other than the lipopolysaccharide O-chain in the immune response to brucellosis.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. I. Examination of field cases

Thirty cows presenting with lameness and persistent serological reactions to Brucella abortus had chronic granulomatous arthropathy of the femorotibial and occasionally other joints. Attempts to culture Brucella or other pathogens gave negative results but organisms of Brucella morphology were seen in fluorescent antibody-stained cryostat sections of synovial tissue. The synovial fluids contained high titres of antibodies to B. abortus and Yersinia enterocolitica O:9 and had elevated total protein and immunoglobulin concentrations showing an oligoclonal electrophoretic profile. Immune complexes and rheumatoid factor were detected in some of the fluids.     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

Serologic response to Brucella abortus of cattle and guinea pigs experimentally inoculated with a Yersinia enterocolitica serotype from milk

Ten strains of Yersinia enterocolitica belonging to ten various serogroups isolated from raw milk were inoculated into groups of five guinea pigs and five calves. Y. enterocolitica serotype 0:16 was the only serotype tested that induced an antibody response to Brucella abortus in calves. No anti-Brucella response could be demonstrated serologically in guinea pigs. Activity of the anti-Y. enterocolitica 0:16 calf sera against B. abortus antigen was shown by the tube agglutination test, and by the complement fixation test. The early agglutinating antibody response was partly sensitive to reduction by 2-mercaptoethanol. This sensitivity decreased later in the response. This is the first report of anti-Brucella responses induced by a serotype of Y. enterocolitica other than 0:9; sera from a group of five calves inoculated with 0:9 were tested by the same serological techniques for comparison.     

Experimental infection of dogs with Brucella abortus

Thirteen female dogs, which included eight principals that were fed approximately 4.4 X 10(10) colony forming units (cfu) of Brucella abortus strain 2308 and five sentinels that were housed with the principals, were examined for serologic responses, blood culture, tissue distribution of the organisms and pathologic lesions. Serum samples from each dog were tested on the day of exposure and on post exposure days 5, 7, 10, 14, 21, 28, 35, 42 and 49 for antibodies to B. abortus, using the brucellosis card (BC), standard tube agglutination (STA), 2-mercaptoethanol (ME) and rivanol (RIV) tests. Antibodies were detected in the principals by day 5 and increased through day 21. The STA test was the first to become positive, followed by the BC, ME and RIV tests. After 28 days, the serologic titers receded. From day 14 through day 42, all principals had greater than or equal to 1:50 STA titers. On day 49, seven principals had greater than or equal to 1:50 STA titers and one had a 1:25 STA titer. The sentinels were negative for all tests, except sentinel number 9 which had STA titers ranging from 1:25 to 1:50 on day 14 through day 35. Blood cultures that were obtained from each principal at intervals from one hour after exposure through 49 days were negative. Brucella abortus was isolated from various lymph nodes of the eight principals and from sentinel number 9, which was apparently infected by ingesting brucellae contaminated feces from the principals. Microscopic lesions were not observed in the culture-positive tissues examined.     

Experimental infection of sheep with Brucella abortus

A case of brodifacoum poisoning is described in a six-year-old male Kelpie cross working dog. The clinical features were severe exercise intolerance, haemorrhage from the oral and nasal cavities, dyspnoea and pale mucous membranes. Diagnosis was confirmed by demonstrating an abnormally long whole blood clotting time. The dog was treated successfully by administering 1 litre of whole blood intravenously, intramuscular vitamin K₁ and a three week course of oral vitamin K₃.

Experience at the Massey University Small Animal Clinic and Hospital has indicated that poisoning of dogs with the newer long acting anticoagulant rodenticides is becoming more common.     

Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay

The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p<0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24-72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples.     

Brucella abortus associated with abortion in a sow

Extract

The present owner of the farm on which this case occurred took it over in May, 1957. He bought in 12 heifers in May, 1958. which had not been vaccinated against Brucella abortus. During May to July, 1958, three of his cows (presumably vaccinated as calves) and one of these heifers aborted. In two of these cases (one cow and one of the heifers), the cause was determined to be B. abortus. Five maiden Large White sows were bought during April and May. Although pigs and cows were run in separate paddocks, the cows occasionally passed along a race to which the sows had access. One of these, the sow “Bess”, was clue to farrow on 20 August, but was observed to be aborting on 6 August.     

Kinetics of detection of blastogenic responses of neonatal calves inoculated in utero with tetanus toxoid, killed Mycobacterium bovis, and killed Brucella abortus.

Nine pregnant cows were laparotomized and their fetuses were immunized with tetanus toxoid, killed Brucella abortus, and killed Mycobacterium bovis. Blastogenesis assays and total leukocyte and differential counts were done when the calves were 1, 2, 3, 7, 14, 21, 28, and 60 days of age. Initial blastogenesis responses to antigens, phytohemagglutinin, and concanavalin A were not positive as frequently as were the responses obtained when the calves were 2 to 3 weeks of age. The probability of obtaining a positive response to an antigen was positively correlated with the magnitude of the response, as determined by delayed-type hypersensitivity skin reactions. Leukocyte and differential WBC counts in immunized calves were similar to those of unimmunized calves. The mean leukocyte count for the immunized calves remained near 16,000 cells/mm3; blood obtained in the first few days after birth contained a greater number of neutrophils than lymphocytes, whereas lymphocyte-to-neutrophil ratio gradually approached those of adult cattle, in which lymphocytes predominate.