Effect of milk stasis on Brucella abortus infection of the mammary gland in goats

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.     

Comparison of three culture techniques for the isolation of Brucella abortus from bovine supramammary lymph nodes

Three different culturing techniques were compared and evaluated to determine the most effective method for isolating Brucella abortus from bovine supramammary lymph nodes (SM's). In method I, the SM was sliced in half, and the inner surface was minced finely with a sterile scalpel. The minced surface was spread onto the agar surface of 4 selective media. In method II, the SM was cut into small pieces and placed in a bag with a volume of phosphate-buffered saline equal to the volume of the lymph node. The bag was placed in a laboratory blender and the SM was macerated for 5 min. The tissue suspension was spread with a sterile cotton swab onto the agar surface of 4 selective media. In method III, the SM was processed in the laboratory blender. One milliliter of the suspension was pipetted into a flask of biphasic medium, and 2 ml of the suspension was pipetted into another flask of biphasic medium. A total of 626 SM's from 285 cows were cultured. Brucella abortus was isolated from 149 (52.3%) cows by 1 or more methods. Brucella abortus was isolated from 136 cows by method I. 137 cows by method II, and 86 cows by method III. Nine (3.2%) cows were positive by method I only, 11 (3.9%) cows by method II only, and 2 (0.7%) cows by method III only. The isolation rate for method III was significantly lower than for method I or II. There was no significant difference between methods I and II.     

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 placentitis in the goat inoculated with Brucella abortus. I. Gross and histologic lesions

Pregnant goats were given Brucella abortus intravenously or in uterine arteries, and tissues from the uterus and placentae were examined at various post-inoculation intervals to study mechanisms of placental infection. Placentitis was present by 5 days post-inoculation and abortions occurred within 11 days. B. abortus was identified in placentae by light microscopy and immunoperoxidase techniques. B. abortus was first seen in erythrophagocytic trophoblasts of the placentome. Subsequently, high numbers of B. abortus were seen in periplacentomal chorioallantoic trophoblasts. Trophoblast necrosis, chorioallantoic ulceration, and large numbers of B. abortus in chorionic villi were present in later stages of infection. These results suggest that entry and replication of B. abortus in trophoblasts precede placentome and fetal infection and that trophoblasts are the source of B. abortus for these tissues. Experimental caprine brucellosis closely resembles bovine and ovine brucellosis and it provides a model to study the intracellular development of B. abortus in trophoblasts.     

Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages

Chronic bovine brucellosis is characterized by persistent infection of the mammary gland. The interaction of live Brucella abortus with bovine mammary gland macrophages was studied in vitro. Opsonization of smooth B abortus strain 2308 and rough strain 45/20 was required for phagocytosis by mammary gland macrophages. When opsonized with specific antiserum, strains 2308 and 45/20 stimulated a considerable oxidative burst when phagocytized by mammary gland macrophages. Intracellular survival rates for strain 2308 were significantly higher than those for strain 45/20. After being phagocytized, B abortus localized in phagosomes and phagolysosomes of mammary gland macrophages.     

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.     

Effect of nursing on Brucella abortus infection of mammary glands of goats

Eight 1-year-old, goats were inoculated intravenously with Brucella abortus (B. abortus) on the day of parturition and necropsied at 28 days after inoculation. Four nursed their kids and four did not (milk was not removed from the udders). Tissues and fluids were examined by bacterial isolation, light microscopy, and serologic methods. Nonnursing goats had high titers of brucellae (less than or equal to 10(8) organisms/ml) in milk (brucellae were isolated from four of four udders), had marked enlargement of supramammary lymph nodes, and had lymphoplasmacytic and histiocytic interstitial mastitis. Immunoperoxidase staining revealed that brucellae were primarily in macrophages and neutrophils of the mammary alveolar and ductal lumens and in macrophages of the subcapsular sinuses of the supramammary lymph node. In contrast, nursing goats excreted brucellae intermittently at low concentrations (less than 10(3) organisms/ml) in milk; brucellae were isolated at necropsy from one of four udders; supramammary lymph nodes were not enlarged; and mammary lesions were not seen. Brucellae were detected in more tissues other than the udder, and serum anti-Brucella antibody titers were higher in nonnursing goats than in nursing goats. The present study indicates that the failure to nurse or release milk enhances localization and replication of B. abortus in mammary glands of goats after parturition, and that mammary gland infection may result in increased systemic spread and persistence of brucellae in the host.     

The response of the supramammary lymph node of the sheep to secondary infection with orf virus

The response of the supramammary lymph node of seven sheep to secondary infection with orf virus was examined by cannulating the efferent lymphatic before infecting the drainage area of the node. All animals developed typical orf lesions and responded after an initial lag period with an increase in total cell output paralleled by a rising proportion of lymphoblast cells. Most lymphoblast contained immunoglobulin with a predominance of the IgG class. When cultured, these cells produced measurable amounts of virus-specific antibody. The proportion of cells which stained with a T-cell-specific monoclonal antibody was measured in two of the sheep and found to decrease as the response developed. These data suggest that the nodal response is directed mainly towards the production of virus-specific antibody. The extent of T-cell involvement remains unclear.     

Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus

Nonvaccinated pregnant cows were segregated retrospectively into 2 groups following inoculation with Brucella abortus strain 2308. One group resisted infection (resistant cows) and the other group developed active infections (susceptible cows) and subsequently aborted. Mammary gland macrophages collected from the 2 groups of cows were compared, using in vitro functional assays. In a chemiluminescence assay, mammary gland macrophages from resistant cows produced significantly (P = 0.014) higher oxidative burst activity than did macrophages from susceptible cows. Macrophages from resistant cows had significantly (P = 0.038) greater bacteriostatic activity against B abortus than did macrophages from susceptible cows. Differences in lysosomal enzymatic activity or Fc receptor expression were not observed for macrophages from the 2 groups of cows. Differences in macrophage function may be one factor responsible for natural resistance to Brucella infection in cattle.     

Experimental Brucella abortus infection in pigs

Sixteen pigs inoculated by the intra-conjunctival, intravenous or subcutaneous routes with Brucella abortus Strain 544 developed a short-lived infection usually accompanied by conjunctival and vaginal excretion of the organism for up to 99 days post-inoculation. Serological tests performed by the agglutination, complement fixation, Rose Bengal plate, antiglobulin, immunodiffusion or ELISA procedures with B. abortus antigens disclosed wide variations in the antibody responses of individual animals. In some cases the serological tests were negative even though the animal was shown to be excreting B. abortus. The intradermal test for delayed hypersensitivity to Brucella antigens gave more consistent results, especially when supported by histological evaluation of the skin reactions.     

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.     

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.     

Brucella abortus infection in 14 farm dogs

Fourteen dogs were obtained from 10 farms with Brucella-infected cattle and were studied for periods ranging from 2 to 81 days. At necropsy, Brucella abortus biovar 4 was isolated from all 14 dogs. Mandibular, medial retropharyngeal, tracheobronchial, and mesenteric lymph nodes yielded the highest rate of recovery. Urogenital infection with active shedding was seen in a single aged bitch. Fecal samples (291 from 13 dogs) were B abortus culture negative. Ten dogs monitored serologically over time had standard tube agglutination test titers that fluctuated between 1:50 with incomplete reaction and greater than or equal to 1:200 with positive reaction. At necropsy, the magnitude of seroconversion was not directly related to the number of culture-positive tissues. The maximal duration of infection, based on the time interval between the last possible exposure to infected cattle and recovery of the organism at necropsy, was 464 days. If it were assumed that infection occurred at about the same time as seroconversion in the cattle herd of origin, maximal observed duration of infection was 539 days. The actual maximal duration of infection could not be determined from this study, but would have exceeded the values reported here. The data indicate that dogs have the potential to infect cattle and could pose a threat for longer duration of disease transmission than had previously been assumed. Although the risk of transmission appears small, infection of human beings or cattle associated with Brucella-infected dogs would cause unfavorable political and economic consequences, particularly in low-incidence areas. Removal of contact dogs from infected herds should prevent such transmission.     

Brucella abortus infection in indigenous Korean dogs

Three dogs reared on a dairy farm with a high incidence for Brucella abortus were serologically positive for B. abortus and no other Brucella spp. The identity of the organism was confirmed to be B. abortus by AMOS (abortus melitensis ovis suis)-polymerase chain reaction with specific primers for B. canis. One hundred percent homology of the canine isolate and the bovine pathogen isolated from the farm was demonstrated. The only possible source of infection was infected cattle on the same farm. It is suggested that dogs be routinely included in brucellosis surveillance and eradication programs.     

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.     

Lymphocytes from one side of the bovine mammary gland migrate to the contra lateral gland and lymph node tissue

The four quarters of bovine mammary glands are completely separated and two quarters on each side (right or left) are connected to ipsi lateral supra mammary lymph nodes. It is not clear whether cells infused into the cistern of the mammary gland are capable of migrating to lymph nodes or the general circulation. To examine cell migration, a prescapular lymph node was removed from each of two lactating and three non-lactating dairy cows, and isolated lymphocytes were stained with Hoechst 33342. Autologous stained cells were infused into the mammary gland and then activated by intramammary infusion of zymosan-stimulated serum (source of C5a). After 17 h, Escherichia coli J5 bacterin was infused into the contra lateral mammary gland to mimic infection. After 43 h cows were euthanized and tissue samples (mammary quarters, right and left supra mammary, mesenteric, ileocecal and prescapular lymph nodes, liver and spleen) were collected for microscopic examination as well as flow cytometric analysis. Hoechst stained cells were detected not only in infused quarters, but also in contra lateral quarters as well as in both supra mammary lymph nodes. This indicates that cells infused into the mammary gland migrate to contra lateral tissues and supra mammary lymph nodes.