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.     

Characterisation of the lymph node immune response following Mycoplasma mycoides subsp. Mycoides SC infection in cattle

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.     

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.     

Assessment of in vitro interferon-gamma responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type

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.     

Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction

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.     

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.     

Gamma interferon-producing CD4 T-cells correlate with resistance to Mycoplasma mycoides subsp. mycoides S.C. infection in cattle

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.     

Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC

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.     

Serological survey to determine the occurrence of respiratory Mycoplasma infections in the Polish cattle population

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.     

Molecular epidemiology of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides subspecies mycoides biotype SC strains

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.     

Susceptibility of goats and calves after experimental inoculation or contact exposure to a Canadian strain of Mycoplasma mycoides subsp. mycoides isolated from a goat.

Transmissibility of Mycoplasma mycoides subsp. mycoides infection from experimentally inoculated goats to other goats and calves was studied. Eight goats and six calves were housed in an 18 m2 room. Six of the goats were inoculated endobronchially with strain D44 isolated from a natural case of polyarthritis in Ontario. These six goats died within a week of Mycoplasma septicemia. The two contact goats or the six calves never showed signs of disease and M. mycoides subsp. mycoides was not recovered from these animals. The contact goats and four calves were killed 25 days after exposure. They were all seronegative, M. mycoides subsp. mycoides was not recovered at necropsy and none had pathomorphological changes attributable to this Mycoplasma. The two remaining calves were inoculated endobronchially with 10(9) CFU of strain D44 and observed for 20 days. They never showed signs of disease and did not have significant lesions at necropsy. Both developed a significant serological response to M. mycoides subsp. mycoides, although this organism was not recovered during the experimental period or at necropsy. This study did not provide evidence for transmission of M. mycoides subsp. mycoides from endobronchially inoculated goats to contact goats or calves and endobronchially inoculated calves did not develop pneumonia. This would suggest that the infection of the goat population in Canada with this pathogen would not be a significant threat to the cattle population.     

Detection of Mycoplasma mycoides subspecies mycoides SC in bovine lung and lymph node tissues by culture, sandwich ELISA and polymerase chain reaction systems

Cattle from Northern Portugal, many with pulmonary lesions typical of contagious bovine pleuropneumonia, were investigated for the presence of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), which is the causative agent of CBPP, with several detection tests. Sandwich ELISA that included a culture enrichment stage, and 2 different PCR diagnostic systems were used to detect MmmSC in lung and mediastinal lymph node tissues from these animals. The comparison of typical CBPP pathology with the results of detection revealed that no single one of these methods provided a perfect match to the pathological data. Best performing tests were the PCR with laser induced fluorescence and PCR with pleuroTRAP kit (Chemicon, Australia), which are diagnostic systems based on amplification of genomic MmmSC DNA followed by sensitive detection of the amplified products. These were followed by the broth-enriched sandwich ELISA, which uses a monoclonal antibody specific to the M. mycoides cluster, to capture the antigen.     

Danofloxacin (Advocin) reduces the spread of contagious bovine pleuropneumonia to healthy in-contact cattle

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most important diseases of cattle in Sub-Saharan Africa. The live T1/44 vaccine is normally used for its control but produces only transient protection and gives rise to adverse reactions. The present study evaluated the efficacy of danofloxacin (2.5% Advocintrade mark, Pfizer Ltd.) for the treatment of naturally infected cattle and in the prevention of CBPP transmission to in-contact cattle. Adult cattle, taken from a natural outbreak, were placed into two groups of 10 animals and kept on a research farm in paddocks 50m apart. One group was treated with 2.5mg/kg danofloxacin on days 0, 1 and 2; the other group were saline treated. On day 2, 10 CBPP-free, seronegative cattle were placed in contact with each of the two groups. All cattle were monitored for 3.5 months. No differences were seen in clinical improvement of the CBPP-affected cattle treated with danofloxacin compared with the untreated CBPP-affected cattle with approximately half of each group being withdrawn because of CBPP or showing CBPP lesions at post mortem examination. Clinical scores of the two groups were also similar. However cattle kept in contact with the danofloxacin-treated CBPP-affected animals showed significantly fewer lesions, less mortality and fewer animals were seropositive (P<0.02) and had reduced clinical scores (P<0.001) compared to cattle kept in contact with untreated CBPP-affected cattle. MmmSC was also isolated from fewer contact controls kept with the treated group. These findings could have important implications for the control of CBPP in Africa.     

Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp. mycoides SC

A study was carried out on four adult cattle to assess the pathogenicity of Mycoplasma mycoides subsp. mycoides SC strain T1/44, currently used as a vaccine for the control of contagious bovine pleuropneumonia (CBPP) in Namibia. Post mortem examination 9 weeks after endobronchial inoculation of the vaccine strain to three of the four animals revealed unilateral pleuropneumonic lesions, pleuritis and well-developed sequesters in two of the three inoculated animals and several small sequesters surrounded by pleuropneumonic lesions in the diaphragmatic and apical lobes in one animal. The fourth animal, which was not directly inoculated but was in close contact with the inoculated animals, revealed only an adhesion area of the lung to the ribcage. Serological examination carried out using the complement fixation test (CFT) detected positive titres in all three intubated animals and the indirect CBPP-LppQ-ELISA was positive for two of the three inoculated animals. The contact animal showed no seroconversion. M. mycoides subsp. mycoides SC was isolated from the sequesters of two of the inoculated animals. Isolation of mycoplasmas was not possible from the third inoculated animal due to heavy contamination of the samples by other bacteria, but the presence of M. mycoides subsp. mycoides SC could be evidenced by PCR from clinical samples. The identity of the T1/44 vaccine strain isolated from the sequesters of two animals was confirmed by T1/44-specific PCR analysis and by IS1296 typing using Southern blot. These results clearly show that inoculation of T1/44 vaccine via the endobronchial route can lead to CBPP.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

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.     

丝状霉形体丝状亚种SC型脂蛋白LppQ N末端基因克隆与序列分析

脂蛋白LPPQ是丝状霉形体丝状亚种SC型(MmmSC)非洲株、欧洲株和疫苗株所特有的。LPPQ N末端域具有良好的免疫原性，在牛体内可诱导产生强大、特异、早期、持续的免疫反应。本研究根据已发表的LPPQ基因序列设计引物，用Pyrobest^TM高保真DNA聚合酶从MmmSC HVRI X株中扩增出了LPPQ N末端基因序列，并进行了克隆与序列测定。核苷酸序列比较结果显示，HVRI X株的LPPQ N末端基因序列与国外发表的序列同源性为99．7％，由其推导的氨基酸序列同源性为99，1％，为脂蛋白LPPQ N末端基因体外表达奠定了基础。     

Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony

Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.     

Experimental field trial with an immunostimulating complex (ISCOM) vaccine against contagious bovine pleuropneumonia

The experimental field trial with an immunostimulating complex (ISCOM) vaccine has been an occasion to explore the role of a Th1 response in the pathogenesis caused by Mycoplasma mycoides subsp. mycoides small colony (MmmSC) and in immune protection. The ISCOM complex is known to promote Th1 response. Antibodies to MmmSC were detected by indirect enzyme-linked immunosorbent assay (ELISA) in the vaccinated cattle, although the levels were lower than in a previous study. No antibodies were detected by complement fixation test (CF). After the challenge infection, vaccinated animals developed CF antibody response. They showed significantly reduced mortality compared with controls. However, gross pathological and histopathological score for vaccinated animals was as high as for the non-vaccinated, characterized by a high inflammatory reaction with histopathology dominated by interlobular pneumonia with vasculitis.     

Extended surveillance for CBPP in a free country: Challenges and solutions regarding the potential caprine reservoir

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.     

Strains of Mycoplasma mycoides subsp. mycoides small colony type isolated in China between 1953 and 1960 show close similarity to strains of the Africa/Australia cluster

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.