Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.     

Analysis of cellular responses to Mycoplasma mycoides subsp. mycoides small colony biotype associated with control of contagious bovine pleuropneumonia

A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.     

Pulmonary and serum antibody responses elicited in zebu cattle experimentally infected with Mycoplasma mycoides subsp. mycoides SC by contact exposure

The purpose of the present study was to characterize the Mycoplasma mycoides subsp. mycoides small colony (MmmSC)-specific humoral immune response at both systemic and local levels in cattle experimentally infected with MmmSC, for a better understanding of the protective immune mechanisms against the disease. The disease was experimentally reproduced in zebu cattle by contact. Clinical signs, postmortem and microbiological findings were used to evaluate the degree of infection. Serum and bronchial lavage fluids (BAL) were collected sequentially, before contact and over a period of one year after contact. The kinetics of the different antibody isotypes to MmmSC was established. Based on the severity of the clinical signs, post mortem and microbiological findings, the animals were classified into three groups as acute form with deaths, sub-acute to chronic form and resistant animals. Seroconversion was never observed for the control animals throughout the duration of the experiment, nor for those classified as resistant. Instead, seroconversion was measured for all other cattle either with acute or sub-acute to chronic forms of the disease. For these animals, IgM, IgG1, IgG2 and IgA responses were detected in the serum and BAL samples. The kinetics of the IgM, IgG1 and IgG2 responses was nearly similar between both groups of animals. No evident correlation could thus be established between the levels of these isotypes and the severity of the disease. Levels of IgA were high in both BAL and serum samples of animals with sub-acute to chronic forms of the disease, and tended to persist throughout the entire experimental period. In contrast, animals with acute forms of the disease showed low levels of IgA in their BAL samples with none or very transient but low levels of IgA in the serum samples. Our results thus demonstrated that IgA is produced locally in MmmSC experimentally infected cattle by contact and may play a role in protection against contagious bovine pleuropneumonia.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

Clinical, humoral and IFNgamma responses of cattle to infection with Mycoplasma mycoides var. mycoides small colony and attempts to condition the pathogenesis of the infection

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides var. mycoides small colony (MmmSC), is one of the most important diseases of cattle in Africa. The role of innate or acquired cell mediated and humoral immunity in conferring protection against MmmSC infection has not yet been elucidated. On the other hand, the pathological lesions caused by the aetiological agent have been considered indicative of an immunopathological process. In this study ten na?ve cattle were exposed to in-contact infection with animals infected by intubation with a strain of MmmSC. Clinical signs, antibody response, IFNgamma release and pathological changes at necropsy were analysed and compared with the events following in-contact infection of an equal number of animals kept under daily treatment with cyclosporine for the entire observation period of 84 days. Cyclosporine is a suppressor of the immune response related to the T-cell system. Under the conditions of the experiment, cyclosporine appeared to condition the pathogenesis of CBPP by delaying the events that follow infection, bringing further support to the possibility that the immune response may have an impact on the disease outcome.     

In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials

In vitro susceptibilities of 16 Mycoplasma mycoides subsp. mycoides large colony type field isolates to 15 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials were fluoroquinolones, tetracyclines and macrolides, with MIC values under 2 microg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.     

Inflammatory cytokines and antigen-responsive mononuclear cells in peripheral blood of cattle infected with Salmonella takoradi

To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4+ T cells to CD8+ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4+ T cells in the infected cattle. In contrast, the numbers of gammadeltaT cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6. p<0.01). Serum TNF-alpha activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs. such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-y was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.     

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type

Minimum inhibitory concentrations (MIC) and minimum mycoplasmacidal concentrations (MMC) of the antimicrobials danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin were determined in vitro for 20 isolates of Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP). The majority of strains were most susceptible to tilmicosin, followed by danofloxacin, oxytetracycline, florfenicol and spectinomycin with MIC50 values of 0.015, 0.25, 0.5, 1 and 8 microg/ml, and MMC50 values of 0.06, 0.5, 8, 8 and 16 microg/ml, respectively. However, tilmicosin had poor mycoplasmacidal activity against two recent strains from Portugal. There was no evidence of resistance to danofloxacin in any of the strains.     

Changes in mononuclear peripheral blood cells in cattle with foot-and-mouth disease

The percentages and absolute numbers of mononuclear peripheral blood cells (MNC) were studied in vaccinated (Vac) and non-vaccinated (control) cattle, challenged with foot-and-mouth disease virus (FMDV). All Vac cattle but none of the controls resisted challenge. Cell populations were studied immediately before and one week after challenge, by direct and indirect immunofluorescence, using polyclonal and monoclonal antibodies against different bovine markers. Total B-lymphocytes, as assessed with polyclonal anti-IgG(H+L) antisera, as well as total mononuclear cells, were normal before and after infection, and did not change in Vac or control groups. Before challenge Vac cattle had higher numbers of IL A-29⁺ (a putative marker for null cells or, alternatively, for γδ T-cells) than control cattle. After challenge, in control cattle, the number of total T-cells, BoT4-bearing (helper) T-cells and BoT8-bearing (cytotoxic/suppressor) T-cells were decreased, while IgM-bearing B-lymphocytes, as well as monocyte/macrophage cells were increased. The number of IL-A29-bearing T-cells did not change after infection in either group. After challenge, Vac cattle also showed increased numbers of IgM-bearing B-lymphocytes and monocyte/macrophage cells, whereas T-subpopulations did not change significantly.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

Cytokine secretion by peripheral blood mononuclear cells from cows infected with Mycobacterium paratuberculosis

OBJECTIVE: To compare cytokine secretion patterns of peripheral blood mononuclear cells (PBMC) from healthy cows and cows subclinically and clinically infected with Mycobacterium paratuberculosis. ANIMALS: 5 noninfected cows, 6 cows with subclinical paratuberculosis, and 4 cows with clinical paratuberculosis. PROCEDURE: PBMC were isolated, and concentrations or activities of secreted interleukin (IL)-1, IL-2, IL-6, tumor necrosis factor (TNF), and interferon-gamma (IFN-gamma) were measured after in vitro stimulation of cells with concanavalin A (ConA), lipopolysaccharide (LPS), or a whole-cell sonicate of M paratuberculosis (MpS). Proliferative responses of PBMC were also determined after stimulation with ConA, phytohemagglutinin, pokeweed mitogen (PWM), or MpS. RESULTS: After stimulation with ConA, cells from subclinically infected cows secreted significantly more, and cells from clinically infected cows secreted significantly less, IFN-gamma, compared with cells from control cows. Cells from cows with subclinical paratuberculosis produced significantly more TNF and IFN-gamma in response to MpS than cells from the other 2 groups. Stimulation of PBMC from subclinically infected cows with ConA or MpS resulted in significantly higher proliferative responses, compared with cells from control and clinically infected cows. In contrast, clinically infected cows had significantly higher proliferative responses to PWM than cells from the other 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in T-cell responses to mitogens or MpS was observed in cows clinically infected with M paratuberculosis, compared with subclinically infected cows, suggesting that activated T cells may delay the progression of paratuberculosis.     

Responses of peripheral blood mononuclear cells in calves infected with Theileria sergenti

The role of peripheral blood mononuclear cell (PBMC) in Theileria sergenti-infected calves was studied by various in vitro assay systems. Proliferation of T cells in mixed lymphocyte protozoa culture (MLPC) increased with parasitemia, and the addition of monoclonal antibodies against T. sergenti merozoites in this MLPC enhanced the response. However, the addition of antibody-positive autologous serum resulted in the suppression of the response. Cell-mediated cytotoxicity of PBMC increased after peak parasitemia. This cytotoxicity increased on co-cultivation of PBMC with T. sergenti merozoites, but the addition of autologous serum suppressed the response.     

Bovine virus diarrhoea virus induces in vitro a proliferative response of peripheral blood mononuclear cells from cattle immunized by infection.

The ability of bovine peripheral blood mononuclear cells (PBMC) to mount a proliferative response to bovine virus diarrhoea virus (BVDV) in vitro was examined. After culturing PBMC in the presence of a non-cytopathic strain of BVDV for 6 days, the magnitude of PBMC proliferation was measured as incorporation of radiolabelled thymidine, present during the last 18 h. Live, but not heat-inactivated, BVDV evoked a proliferative response of PBMC obtained from cattle seropositive to the virus. However, PBMC from seronegative or persistently BVDV-infected animals were not stimulated by BVDV. The presence of live BVDV did not alter the proliferative response of PBMC to stimulation with concanavalin A.     

Comparison of complement fixation test,immunoblotting, indirect ELISA,and competitive ELISA for detecting antibodies to Mycoplasma mycoides subspecies mycoides small colony (SC) in naturally infected cattle from the 1995 outbreak in Botswana

Four serological tests were compared in order to evaluate their efficacies in detecting antibodies to M. mycoides subspecies mycoides SC in cattle sera sampled in 1995 from herds affected with contagious bovine pleuropneumonia (CBPP) in the north-western part of Botswana. Tests that were compared included immunoblotting test (IBT), indirect enzyme-linked immunosorbent assay (i-ELISA), competitive enzyme-linked immunosorbent assay (c-ELISA) and complement fixation test (CFT). The percentages of seropositive samples in the iELISA (48%) and in the c-ELISA (48%) were similar but were comparatively lower than those obtained by the IBT (57%) and CFT (61%). The percentages of positive sera in the IBT and CFT were also similar and overall the efficacy of these tests was better than that of the two ELISA tests. There was 95.5% agreement between the IBT and CFT, 85% agreement between the IBT and c-ELISA, 90.9% agreement between the IBT and i-ELISA, 88.6% agreement between the i-ELISA and CFT, 80% agreement between the c-ELISA and CFT and 90% agreement between the two ELISA tests. It became clearly evident from this comparative study that no single serological test was capable of detecting all animals affected by CBPP under natural field conditions of infection.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation.