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1.
Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.  相似文献   

2.
Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.  相似文献   

3.
Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.  相似文献   

4.
Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease of cattle and buffalo caused by Mycoplasma mycoides subsp. mycoides "Small Colony" (MmmSC). The agent of CBPP has been isolated from goats in different countries including CBPP-free areas. Goats can therefore be regarded as a putative MmmSC reservoir. No diagnostic test for CBPP surveillance in goats has been proposed as yet. Furthermore, serological tests could be seriously hampered by a widespread caprine infection due to the subspecies M. mycoides subsp. capri (Mmc), which is antigenically very close to MmmSC and displays high levels of genetic variability. A competition ELISA (cELISA) is currently used to screen for CBPP in cattle at the herd level in infected areas. The aim of this study was to see if the same cELISA would be specific enough to be used to screen goats despite the potential concomitant infection with Mmc. The cELISA titers of goats from Mmc-infected and non-infected herds were comparable and negative using the accepted cutoff for bovine sera. In contrast, seroconversion was observed in goats experimentally inoculated with an Mmc strain that cross-reacted with a monoclonal antibody targeting the same epitope as that used in cELISA. The probability of such false positivity occurring under field conditions is very low since Mmc strains with such an atypical antigenic profile emerge only rarely as a result of random nucleotide variation of the epitope-coding region. In conclusion, the commercially available cELISA can be considered specific enough to be used as a primary test to monitor passage of the CBPP agent in goats, but its sensitivity in goats requires further investigation.  相似文献   

5.
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.  相似文献   

6.
Contagious bovine pleuropneumonia (CBPP) is a lung disease caused by the bacterial pathogen Mycoplasma mycoides ssp. mycoides small colony type (MmmSC). It has been spreading due to a number of factors including poor vaccine efficacy and poor sensitivity of current diagnostic tests. The purpose of this study was to assess interferon gamma (IFN-gamma) release after stimulation of peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. PBMC collected from 15 artificially infected animals were incubated with different concentrations of total MmmSC antigen. After 72h of incubation the IFN-gamma release was measured and found to be elevated in 11 animals. We did not observe a correlation between IFN-gamma release of animals with and without pathomorphological gross lesions. Therefore, our data do not confirm a role for CD4 T-lymphocytes in protection, since there is no correlation between IFN-g secretion (supposed to be mainly derived from CD4 T-cells) and disease severity. Additionally, we applied immunocytochemistry on affected lung tissue and detected no build up of T-lymphocytes (CD4 T-cells, CD8 T-cells) but a high presence of myeloid cells.  相似文献   

7.
In this study, six Chinese strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated between 1953-1960 were analysed and their molecular characteristics compared to those of the African PG1 and Afade strains, the European C305 and 138/5 strains and the closely related caprine M. mycoides subsp.mycoides large colony type Y-goat strain. PCR amplification of long DNA fragments showed that the six Chinese strains, the PG1 strain and the Y-goat strain, just like Afade, did not have the 8.84 kb deletion characteristic of the European strains C305 and 138/5. In comparison, the lppB gene sequence of the six MmmSC Chinese strains was found to be 99% homologous to that of PG1and Afade, but <93% homologous to the Y-goat sequence. The anti-rLppB antiserum reacted with PG1, Y-goat and the six Chinese strains at 67 kDa sites in Western blot, indicating that the lppB gene and its encoding protein exist in the Chinese strains. Multilocus sequence analysis (MLSA) of MmmSC strains from various regions confirmed that the Chinese strains were identical to the African and Australian cluster. This finding was further supported by the outcome of selective primer amplification. Based on these results, it is suggested that CBPP in China may have originated from Australia.  相似文献   

8.
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.  相似文献   

9.
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most significant cattle disease in Africa. The control measures, which led to eradication from numerous countries are not feasible in Africa where the only prophylaxis relies on vaccination. However, the attenuated vaccines, used up to now in Africa, are of low efficiency. The development of an improved vaccine is, therefore, a necessity. The purpose of this study was to compare some immunological parameters in MmmSC-infected cattle (endobronchial versus natural in-contact infection) and assess the response in correlation with the clinical outcome (death versus recovery). Characterization of the immune parameters elicited in recovered animals, known to be refractory to new infection, will be an important step towards development of new vaccines against CBPP. A significant outcome of this study was the demonstration that all MmmSC-infected cattle developed a MmmSC-specific cell-mediated immune response. A kinetic analysis of the MmmSC responsiveness showed that the main difference between endobronchially- and in-contact infected animals was the delay before the onset of the MmmSC-specific immune response. The first MmmSC-responding PBMC sample was selected from each animal for cell phenotyping. The phenotypic analysis of this early MmmSC-induced response revealed the predominant contribution of the CD4 T-cells in all animals whereas IFNgamma was only constantly produced in recovered animals. Evolution of this early MmmSC-specific immune response was then followed by a kinetic analysis of the MmmSC-induced CD4 T-cell response and IFNgamma released. The results demonstrated that in recovered animals, the MmmSC-specific CD4 Th1-like T-cell response was maintained until slaughtering whereas in animals with acute disease, progression of CBPP was associated with a decreased ability of the PBMC to produce IFNgamma. The results led to the identification of immune parameters, which correlate with protection against CBPP and to a relevant strategy for the development of improved vaccines against this disease.  相似文献   

10.
Mycoplasma mycoides subsp. mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). Although eradicated in most developed countries, the disease reappeared in Europe in the 1990s. This reappearance may have been caused either by importation from sub-Saharan Africa, where CBPP is still endemic, or by the reemergence of virulent strains in Europe, as suggested by earlier studies. A multilocus sequence analysis scheme has been developed to address this issue and, most importantly, to be able to monitor new epidemics. The alignment of the full genome sequence of the reference strain PG1 and the partial genome sequence of a pathogenic strain allowed the identification of polymorphic sites. Nineteen initial loci were selected within housekeeping genes, genes of unknown function and non coding sequences. The suitability of these loci for genotyping MmmSC strains was first tested on six strains of diverse geographic origin. The analyses showed that the published PG1 sequence contained a number of specific polymorphisms that were therefore of no use for molecular typing. Among the eight informative polymorphic loci finally selected, only one (ftsY) was positioned within a housekeeping gene. Three main groups and 31 different allelic profiles were identified among 51 strains and strain variants examined. Cluster analysis confirmed that European strains from the 1990s did not originate from Africa. It also showed a genetic link between a European strain isolated in 1967 and those found in southern Africa and Australia. This was in agreement with historical data showing that CBPP was introduced in these regions during colonisation in the 19th century.  相似文献   

11.
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.  相似文献   

12.
An indirect ELISA, based on the specific and strongly antigenic recombinant peptide of the N'-terminal half of the lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides small colony type (SC) was developed for the detection of antibodies to M. mycoides subsp. mycoides SC. It was evaluated for its suitability for serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP). The recombinant peptide containing poly-histidine residue tails was expressed in Escherichia coli and subsequently purified by Ni(2+) chelate affinity chromatography to be used as antigen to coat microtiter ELISA plates. The specificity of the antigen was tested against rabbit hyperimmune sera directed against related Mycoplasmas of the M. mycoides cluster and with sera from cattle that were either free of CBPP, but suffered from other mycoplasmal infections such as M. bovis, or showed cross-reactions in the complement fixation test. The sensitivity of the ELISA was assessed with sera from artificially infected animals and with sera from cattle originating from areas where CBPP was endemic at the time of blood sampling. The study revealed that the ELISA was both specific and sensitive for CBPP positive bovine sera and was shown also to be robust to harsh climatic conditions.  相似文献   

13.
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.  相似文献   

14.
AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.  相似文献   

15.
Mycoplasma bovis and Mycoplasma mycoides subspecies mycoides small colony (MmmSC) are causes of bovine mycoplasmosis and contagious bovine pleuropneumonia (CBPP), respectively, and are responsible for serious economic losses in cattle around the world. CBPP was last reported in Poland in 1939 but bovine mycoplasmosis is believed to be endemic. A survey of 3670 serum samples for antibodies to M bovis and MmmSC from 361 herds in 16 Polish provinces Poland between 2007 and 2010 found no evidence of CBPP. The seroprevalence of M bovis, however, appeared high with 76.7 per cent of samples giving a positive reaction in the ELISA test, which did not appear to reflect the clinical disease status of the cattle. Adjusting the sensitivity of the test reduced the prevalence to 28.2 per cent and reflects the levels reported in other European countries.  相似文献   

16.
Contagious bovine pleuropneumonia is a bacterial disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), and included in list A of the Office International des Epizooties. It is one of the major constraints to cattle raising in sub-Saharan and south-western Africa and also a threat to all countries currently free of the disease. MmmSC strains were considered very homogeneous until 1995, when various techniques such as enzymatic restriction of whole DNA or Southern blotting showed that this was not the case. These techniques are unfortunately difficult to standardize and require the extraction of DNA from an MmmSC culture. We therefore decided to investigate the possibility of constructing a molecular epidemiology tool based on multilocus sequence analysis (MLSA) with PCR amplification of various loci followed by sequencing. Six loci were found suitable for this purpose and an additional PCR was designed to detect the presence of an 8.8kb deletion described by others in some strains. Fifteen different MLSA profiles were evidenced in our study. They allowed a clear distinction between European, south-western African and sub-Saharan strains. In addition, the results obtained on strain PO1967 confirmed its European origin, even though it does not exhibit the 8.8kb deletion. This new tool for contagious bovine pleuropneumonia may prove particularly useful for identifying MmmSC strains in countries at risk from contamination. It can also easily be refined by adding more strains or other loci of interest.  相似文献   

17.
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), is still a major cattle disease in Africa. Development of long-term protective vaccines, the only relevant strategy to achieve CBPP eradication, requires the characterisation of the protective immune mechanism. To this aim, the present study investigated the cellular immune response persisting in the lymph nodes of cattle infected naturally and experimentally by contact, one year post exposure. The lymph node cell composition, MmmSC responsiveness and phenotype of the MmmSC-responding lymphocytes were compared between animals according to the different outcomes of the infection. To unravel the protective mechanism, the study focussed on the MmmSC-specific memory immune response generated in recovered cattle, known to develop long-term immunity and to be resistant to reinfection. An MmmSC-specific immune response, mediated by IFNgamma-secreting CD4 T-cells, was detected in the lymph nodes of all recovered cattle. Furthermore, the magnitude of this immune response was significantly higher in animals with complete recovery than in recovered animals presenting lung sequestra. The findings suggest that, in recovered cattle, a subset of MmmSC-primed IFNgamma-secreting CD4 T-cells homed to the regional lymph nodes as MmmSC-specific memory T-cells, likely responsible for the protective anamnestic response. Induction and expansion of this subset of MmmSC-specific CD4 memory T-cells might be a major goal to develop efficient long term protective vaccines against CBPP.  相似文献   

18.
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.  相似文献   

19.
收集了6个来自不同地方中国历史流行的丝状支原体丝状亚种SC型（Mycoplasma mycoides subsp．MycoidesSC，MmmSC）毒株与PG1、Y—goat进行分子特征比较。全茵体蛋白电泳与PG1完全相同，而与Y-goat不同；PCR结果显示6个中国分离株与PG1、Y-goat都没有缺失8．84kb片段；LppB全基因序列比较发现与PG1同源性达99％，而与Y—goat同源性很低；多位点基因序列类型（Multilocus sequence types，MST）分析显示与非洲澳大利亚群完全相同。以上证据显示中国CBPP流行株应归属于非洲澳大利亚群。  相似文献   

20.
Contagious agalactia is an ovine and caprine mycoplasmosis which manifests as mastitis, arthritis and keratoconjunctivitis. Mycoplasma agalactiae is recognised as a causal agent but M mycoides subspecies mycoides (LC), and M capricolum may also be responsible for this syndrome in goats. The clinical signs are not pathognomonic; diagnostic procedures are based on isolation of the organism from diseased animals or by detection of seroconversion. An ELISA specific for M agalactiae and M m mycoides (LC) is described. The specificity of the antigens was demonstrated by immunoblotting and by ELISA using monospecific hyperimmune rabbit sera. A correlation of ELISA activity with other serological tests and isolation of mycoplasmas was carried out in two goat herds under field conditions. Results indicate the ability to detect subclinical mycoplasma infection and individual carrier goats on the basis of ELISA, a finding which will assist control procedures.  相似文献   

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