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.     

Identification and differentiation of European and African/Australian strains of Mycoplasma mycoides subspecies mycoides small-colony type using polymerase chain reaction analysis.

Mycoplasma mycoides subspecies mycoides small-colony type (M. m. m. SC) is the cause of the economically important contagious bovine pleuropneumonia. Isolates from Africa and Australia have previously been documented to have a fragment of approximately 8.84 kb, which is absent in European strains. A set of polymerase chain reaction (PCR) primers over this region was designed to identify M. m. m. SC isolates and separate European strains from those of Africa/Australia. Specificity of the PCR assay was achieved through the positioning of an oligonucleotide within the insertion sequence IS1296, upstream of this deletion, which then was paired with a reverse primer, upstream of the deletion, within the 8.84 kb-deleted region or downstream of the deletion, generating fragments of 1.1 kb (all M. m. m. SC strains), 1.4 kb (African/Australian strains only) and 1.3 kb (European strains only), respectively. Identification and differentiation was specific for DNA from M. m. m. SC with no amplification of DNA from other cluster members or closely related species. The PCR products did not require differentiation by use of a restriction endonuclease, and have potential for use in detection of this organism in clinical samples.     

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.     

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

Lipoprotein lppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP). The lppC gene was found in the type strain of M. mycoides subsp. mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M. mycoides subsp. mycoides SC, of which only one seems to be functional. Genes homologous to lppC have also been detected in closely related mycoplasmas such as M. mycoides subsp. mycoides large colony (LC) type and in M. sp. bovine group 7. lppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II. The amino acid sequence of lppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M. mycoides subsp. mycoides SC. The N-terminal domain of the mature lppC seems to be surface exposed. The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane. A recombinant peptide representing the N-terminal half of lppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts. The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M. mycoides subsp. mycoides SC.     

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.     

Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC.

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.     

Phylogeny of the Mycoplasma mycoides cluster as shown by sequencing of a putative membrane protein gene

The Mycoplasma mycoides cluster is made of six species that are closely related both genetically and phenotypically. Two are of particular importance, M. mycoides subsp. mycoides SC causing contagious bovine pleuropneumonia and M. capricolum subsp. capripneumoniae causing contagious caprine pleuropneumonia. The sequences of a putative membrane protein gene and partial flanking open reading frames have been obtained from various strains in this cluster, including all reference strains. Sequence analysis showed this locus is present and fully conserved in all strains of M. mycoides subsp. mycoides SC isolated from geographically most distant places worldwide. In M. capricolum subsp. capripneumoniae polymorphism in this locus has been found at seven positions and revealed that they can be used as epidemiological markers. Conserved regions were used to define a primer pair that enables the amplification by PCR of two fragments 302 and 1298bp long, respectively. The 302bp long fragment contains an intergenic sequence that can be used for phylogenetic studies or for identification purposes. Parsimony analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides cluster into two subgroups that is in accordance with results obtained by phenotypic methods. Two lineages exist within the capricolum subgroup, one of them clustering strains identified as M. capricolum subsp. capricolum, M. capricolum subsp. capricolum and M. sp Bovine Group 7. However M. capricolum subsp. capripneumoniae strains can readily be identified by three specific nucleotide positions or by sequencing the 1298bp long fragment. There is no clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides subsp. mycoides LC and M. mycoides subsp. capri should not be separated into two subspecies. Mycoplasma mycoides subsp. mycoides SC strains can easily be distinguished as they bear an insertion sequence 15bp downstream from the stop codon of the membrane protein gene.     

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.     

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.     

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.     

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.     

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.     

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.     

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.     

丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

本研究旨在建立丝状支原体簇和多杀性巴氏杆菌的双重PCR检测方法,从而为临床上同时检测这2类病原的感染提供一种更方便、快捷、准确的工具。本研究采用2对特异性检测丝状支原体簇和多杀性巴氏杆菌的引物,对PCR反应体系和反应条件进行了优化,并对双重PCR的特异性及敏感性进行了评价,随后采用该方法对52份临床样本进行了检测。结果显示,所建立的双重PCR方法能同时扩增丝状支原体簇成员和多杀性巴氏杆菌的DNA,而对来源于其他常见病原的DNA均无扩增;对丝状支原体簇和多杀性巴氏杆菌的最低检测限分别为24.8和28.9 pg;能成功地从临床样本中检测丝状支原体簇成员和多杀性巴氏杆菌。结果表明,本研究所建立的双重PCR方法具有很好的特异性和敏感性,为临床丝状支原体簇和多杀性巴氏杆菌感染的快速诊断、病原鉴定及流行病学调查提供了有效的方法。     

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.     

Rapid screening of H(2)O(2) production by Mycoplasma mycoides and differentiation of European subsp. mycoides SC (small colony) isolates

Mycoplasma mycoides strains were screened for the ability to produce H(2)O(2) from glucose and glycerol metabolism using rapid and simple colorimetric assays. In quantitative assays, H(2)O(2) production by washed cell suspensions was detected by the oxidation of o-dianisidine in the presence of peroxidase. In qualitative assays, a 3,3'-diaminobenzidine-peroxidase reagent was applied to colonies on agar plates. Both methods enabled differentiation of European subsp. mycoides SC (small colony) isolates from other M. mycoides strains by their inability to produce H(2)O(2) from glycerol metabolism. In addition, two strains of subsp. capri were identified which produced large amounts of H(2)O(2) from glucose oxidation. In lysed cells of these strains, NADH oxidation gave approximately 1 mol H(2)O(2) per mol NADH oxidised whereas in 36 subsp. mycoides and 10 other subsp. capri strains, the quantity produced was 0.01-0.20mol H(2)O(2) per mol NADH oxidised.     

丝状支原体丝状亚种SC生物型PCR检测方法的建议

建立了一个可以识别丝状支原体丝状亚种SC生物型（MmmSC)的PCR方法。根据丝状支原体丝状亚种SC生物型（MmmSC)相关核苷酸序列，设计合成了MC1、MC2和SC1、SC2两对引物。MC1、MC2是一对簇特异性引物，用于鉴别丝状支原体族6个成员，对MmmSC、MmmLC扩增出与预期结果相符的462bp片段；而SC1、SC2是针对MnnSC的一对特异性引物，只能对MmmSC扩增出277bp的片段，经过VspI、Bsp143I和Dral三种限制性内切酶鉴定与预期结果相符，而不能扩增出MmmLC型Y－goat代表株，说明具有非常好的特异性。对MmmSC进行的敏感性试验显示SC1、SC2引物能够检测到100个CFU，具有非常高的敏感性。     

牛支原体、无乳支原体和丝状支原体丝状亚种小克隆三重PCR检测方法的建立及初步应用

本研究旨在建立一种可一次性区分牛支原体、丝状支原体丝状亚种小克隆和无乳支原体的三重PCR诊断方法,为临床诊断和流行病学调查提供可靠检测技术.根据GenBank发表的上述3种病原的基因组序列保守区域设计3对特异性引物建立三重PCR方法;确定其检测敏感性,以猪支原体、鸡支原体、无乳支原体和丝状支原体丝状亚种小克隆基因作模板检验其特异性;同时和病原分离鉴定结果对比其准确性.结果表明在优化体系和条件下能够同时得到扩增长度为448、549、375 bp 3条特异性片段,未扩增出猪、鸡支原体模板特异性片段;其敏感性(可检测到的最小模板DNA含量)为0.8 ng·μL-1;36份临床样品检测结果显示,三重PCR检测结果与分离培养鉴定方法一致,均能鉴定出牛支原体阳性病料.本研究建立的三重PCR诊断方法能够一次性鉴别3种支原体,具有高敏感性、特异性和准确性,可用于临床诊断和流行病学调查.     

Use of an ELISA for differential diagnosis of Mycoplasma agalactiae and M mycoides subspecies mycoides (LC) in naturally infected goat herds.

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.