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.     

Experimental fetal infection with bovine viral diarrhea virus. II. Morphological reactions and distribution of viral antigen.

The effect of an infection with bovine viral diarrhea virus on fetal bovine tissues as well as the tissue-localization of viral antigen are described. Four bovine fetuses, 120-165 days of gestation, were inoculated in utero with a second passage virus strain. Lymphoid tissues were studied by light and electron microscopy. The infection induced precocious development of the secondary lymphoid organs. Characteristic changes were seen in postcapillary venules, cells of the mononuclear phagocyte system and lymphoid follicles. In all four fetuses the thymus was hypoplastic. In three fetuses this implicated a morphological immaturity, but no actual pathological alterations. In the fourth fetus, the hypoplasia was caused by necrosis and depletion of lymphocytes, attended by infiltration of macrophages. Histopathological changes were also noted in the cerebellum of three fetuses, consisting of necrosis in and depletion of the external germ layer and in the skin and mucous membranes of all four fetuses. The viral antigen was present in cells of the mononuclear phagocyte system, primarily in the lymphoid tissues. The infection was further demonstrated in the affected cerebelli.     

Cellular and humoral responses of Brucella abortus-infected bovine fetuses

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.     

Histopathologic findings in Brucella abortus-infected, pregnant goats

Twenty-eight pregnant goats in midgestation were exposed to a bovine pathogenic strain of Brucella abortus to determine the histologic changes associated with infection. Does were necropsied 0 to 7 days or 4 to 6 weeks after delivery of aborted, stillborn, or live, full-term fetuses. Aborted and stillborn fetuses were necropsied within 16 hours of delivery. Selected, paired tissue specimens were collected for histologic and bacteriologic examination. An avidin-biotin-peroxidase complex immunostaining procedure was used to detect Brucella antigen in tissue section. Histologic changes were evident in specimens from infected does and aborted fetuses. Postpartum does had endometritis, lymphoid hyperplasia in lymph nodes and spleen, and lymphocytic mastitis. The most prominent finding in aborted fetuses was an interstitial pneumonia. Lesions in does and fetuses were similar to those described in Brucella-infected cows and fetuses; however, lesions were less consistently observed in goat fetuses than has been observed in bovine fetuses. Brucella antigen was detected by immunoperoxidase staining within the cytoplasm of placental chorioallantoic trophoblastic cells, macrophages, neutrophils, and uterine epithelial cells. Also, stained brucellae were free in placental and fetal vascular lumens and in the interstitium of autolyzed fetal tissues.     

Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.     

Evaluation of serologic and cellular immune responses of cattle to a nonlipopolysaccharide antigen from Brucella abortus

Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3. Sera from strain 19-vaccinated cows did not have detectable amounts of Ab. Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows. There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows. Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen. Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes). Monocytes obtained from any group did not bind NLA. Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding. Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.     

A comparative pathological study on canine necrotizing meningoencephalitis and granulomatous meningoencephalomyelitis

Canine necrotizing meningoencephalitis (NME) and granulomatous meningoencephalomyelitis (GME) were compared pathologically. Gross observation exhibited lateral ventricular dilation and discoloration, malacia and/or cavitation of the cerebrum in NME. On the contrary, gross changes were milder in GME, except for occasional visible granulomatous mass formation. Histopathologically, the lesions of NME were distributed predominantly in the cerebral cortex and various degrees of inflammatory and necrotic changes were observed according to clinical stages. Besides, microscopic lesions of GME were mainly distributed in the white matter of the cerebrum, cerebellum and brainstem, which are characterized by perivascular cuffing, multiple granulomas and leptomeningeal infiltrates. Although macrophages and lymphocytes were predominant in the inflammatory lesions of both disorders, macrophages in GME transformed into epithelioid cells and exhibited more massive infiltration. Although lectin RCA-1-reactive cells were numerous in both disorders, lysozyme immunoreactive cells in NME were fewer than that in GME. Parenchymal infiltration of MAC387-positive cells was common in GME and limited in NME. The number of CD3-positive lymphocytes in the GME lesions tended to be greater than in NME, though the difference was not statistically significant. Morphological and immunohistochemical differences of the lesions, in particular, the characteristics of infiltrative macrophages may reflect these different pathogeneses of the two disorders.     

Kinetics of in vitro bovine lymphocyte immunostimulation with a Brucella abortus antigen.

A Brucella abortus-soluble antigen was investigated, using in vitro assay of lymphocyte immunostimulation, to determine which concentration of this antigen and which period of incubation of the lymphocyte cultures would induce maximum specific lymphocyte immunostimulation as an additional method for further study of B abortus infection in cattle. Soluble antigen was prepared from autoclaved cells of B abortus strain 1119-3. Peripheral blood lymphocytes were obtained from cattle infected with B abortus and from healthy control cattle not infected with B abortus. The lymphocytes were prepared by the Ficoll-Hypaque density gradient technique, suspended in RPMI 1640 medium (1.5 X 10(6)/ml), cultured with several dilutions of soluble antigen, and incubated. Prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine and, after harvesting, assayed for [3H]thymidine incorporation in DNA by a liquid scintillation spectrometer. Maximum specific immunostimulation of lymphocytes from B abortus-infected cattle was induced in this assay system with 6 days' incubation and 22 microgram of protein/ml/1.5 X 10(6) lymphocytes, using protein content to express concentration of soluble antigen in this system.     

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.     

Levamisole potentiation of antigen specific lymphocyte blastogenic response in Brucella abortus exposed but nonresponsive cattle

Incubation of Brucella abortus (field strain) infected and strain 19 vaccinated bovine peripheral blood lymphocytes with B. abortus antigen and levamisole caused a consistently significant increase in [3H] thymidine uptake when compared to cultures without levamisole. Levamisole did not potentiate B. abortus-induced blastogenic response of lymphocytes from non-exposed cattle. A dose response study showed that 10 micrograms/culture induced maximum potentiation of B. abortus-induced lymphocyte stimulation. Using the 10 micrograms/well concentration of levamisole, further studies were conducted to determine the net potentiation of the blastogenic responses in lymphocytes from B. abortus (field strain) infected cattle. B. abortus strain 19 vaccinated but nonresponsive and non-exposed cattle. Levamisole significantly potentiated the B. abortus-induced lymphocyte blastogenesis in lymphocytes from unresponsive cattle.     

Bovine ileal dome lymphoepithelial cells: endocytosis and transport of Brucella abortus strain 19

Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitium and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propria contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

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.     

Immunohistochemical characterization of tumor cells and inflammatory infiltrate associated with cutaneous melanocytic tumors of Duroc and Iberian swine

The immunophenotype of tumor cells and inflammatory infiltrate associated with cutaneous melanocytic lesions (29 melanocytomas, two malignant melanomas, and 23 residual lesions) from 54 adult Iberian and Iberian x Duroc pigs were examined using a panel of nine antibodies. All neoplastic cells were vimentin+, cytokeratin-, and alpha-1-antitrypsin- and the majority were S100+, whereas all pigmented macrophages were vimentin+, cytokeratin-, and S100- and most expressed alpha-1-antitrypsin. Regressing tumors were characterized by zones with low density of neoplastic cells accompanied by heavy infiltration of CD3+ T lymphocytes, whereas zones with high density of neoplastic cells showed very low numbers of CD3+ T lymphocytes. The infiltrate of CD79a+ B cells and IgG, IgM, and IgA plasma cells was low. The majority of lymphocytes of the peri- and intratumoral infiltrate were major histocompatibility complex class II+, but neoplastic cells did not express class II antigen. The 17 residual lesions examined were composed of macrophages containing abundant melanin pigment and low to moderate numbers of CD3+ T lymphocytes. The results of the present study suggest that the local cellular immune response plays a crucial role in the host response that induces regression of cutaneous melanomas and melanocytomas of the Iberian and crossbred Iberian x Duroc pigs.     

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus.

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.     

Immunomodulation in cattle immunized with Brucella abortus strain 19

Regulation of the bovine immune response to immunization with Brucella abortus Strain 19 (S19) was investigated through application of a modification of an assay to measure suppressor T lymphocyte activities in humans and through development and characterization of antigen-stimulated T lymphocyte lines in vitro. A total of nine of steers were alloted into two groups: control (n = 4) and S19-immunized (n = 5). Peripheral blood mononuclear cells (PBMC) from each animal were cultured in vitro with mitogens (concanavalin A (Con A) and pokeweed mitogen (PWM], B. abortus antigens (B. abortus soluble antigen (BASA) and whole heat-killed B. abortus cells (HKC)) and media alone periodically from days 4 through 49 of the experiment. Supernates from these cultures were assayed for immunomodulatory activity(s) by addition to indicator cultures stimulated with suboptimal concentrations of Con A. Supernates from PBMC of S19-immunized steers generated with B. abortus antigens significantly (P less than 0.05) suppressed indicator cell responses as compared to those from control steers on days 35 and 49 post-immunization. This suppressive activity from PBMC of immunized cattle with respect to that of control cattle could also be induced through mitogenic stimulation with Con A or PWM. On day 49 of the study, suppressive activity was spontaneously released from the PBMC of immunized cattle. T lymphocyte lines were initiated from two S19-immunized steers at 2 and 9 weeks post-immunization. These T cell lines were characterized with respect to proliferative responses to B. abortus antigens through in vitro assay and surface marker expression through indirect immunofluorescence with a limited panel of monoclonal antibodies. Results from the present study indicated that S19 immunization induces a subpopulation(s) of cells in the PBMC of cattle capable of regulating the in vitro response to B. abortus. This regulatory activity is detectable by in vitro assay as early as 7 weeks post-immunization. Furthermore, the regulatory cell(s) appear to involve BoCD8+ T, lymphocytes which are specific for B. abortus antigens.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

In situ characterization of mononuclear leukocytes in skin and digestive tract of persistently bovine viral diarrhea virus-infected clinically healthy calves and calves with mucosal disease

The phenotypic identity of mononuclear leukocytes in skin and digestive tract of bovine viral diarrhea virus (BVDV)-infected animals was examined using sensitive single and double labeling immunocytochemical techniques. Occurrence and distribution of B lymphocytes, T lymphocytes of either the helper (BoT4) or suppressor/cytotoxic (BoT8) subsets, and macrophages (M phi) were examined. No differences were found in the skin and digestive tract of persistently infected clinically healthy calves and uninfected controls, despite widespread virus-antigen presence in keratinocytes (stratum basale and stratum spinosum) of skin and upper digestive tract, in dermal/subepithelial macrophages (M phi), in gut-epithelial cells, and in lymphocytes and M phi/monocytes of blood and lymphoid tissues of the former group. M phi and Langerhans cells (LC) were the prevailing leukocyte types in the keratinized epithelia and subepithelial connective tissues, with occasional T lymphocytes (mostly intraepithelially located and of the BoT8 phenotype). No B cells were seen. Some infiltrating leukocytes also contained virus antigen. In animals succumbing to mucosal disease (MD), hyper- and parakeratotic changes, as well as necrotizing epithelial lesions, were accompanied by massive infiltration in dermis and epithelium of major histocompatibility complex (MHC) class II antigen positive cells (M phi and LC), some T lymphocytes (predominantly BoT8 positive cells), and, only rarely, B cells. M phi also predominated in lamina propria of the gastrointestinal tract. Findings suggest that M phi activation is an important aspect of MD pathogenesis. In contrast, the contention that T lymphocytes may play a major role could not be substantiated.