A specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP)

Contagious caprine pleuropneumonia is a severe infectious disease of goats in Africa and the Middle East. It is caused by a fastidious mycoplasma, Mycoplasma capricolum subsp. capripneumoniae, a member of the "M. mycoides cluster". Members of this cluster share genomic and antigenic features, which result in common biochemical and serological properties, complicating species identification. Two species of this cluster, M. mycoides subsp. capri and M. mycoides subsp. mycoides large colony biotype, are very often isolated from clinical cases resembling contagious caprine pleuropneumonia. Furthermore, in the laboratory, M. capricolum subsp. capripneumoniae can be easily confused with the closely related capricolum subspecies. Considering these constraints and the scarcity of available methods for identification, a specific polymerase chain reaction was developed. A DNA fragment of 7109 bp containing genes coding for the arginine deiminase pathway (ADI) was chosen as target sequence for the selection of a specific primer pair. The full ADI operon from M. capricolum subsp. capripneumoniae strain GL100 was sequenced. Polymorphism within this locus was analyzed by comparison with the sequence from the closely related IPX strain (M. capricolum subsp. capricolum). It varied from 0.6% to 3.5%. The highest divergence was found in a region coding for arcD. Therefore, this gene was chosen as target for the specific amplification of a 316 bp-long DNA fragment. The specificity of this PCR was validated on 14 M. capricolum subsp. capripneumoniae strains and 27 heterologous strains belonging to the "M. mycoides cluster" and M. putrefaciens. This new PCR will be a valuable tool for the surveillance of contagious caprine pleuropneumonia.     

Mycoplasmas isolated from the respiratory tract of cattle and goats in Tanzania

A microbiological study of the mycoplasma flora in the respiratory tracts of cattle and goats in selected regions of Tanzania is described. In the examination of cattle, mycoplasmas were isolated from 60 (17.8%) of the 338 examined lung samples, 8 (47.1%) of the 17 lymph nodes, 4 (13.3%) of the 30 pleural fluid samples and 4 (3.9%) of the 103 nasal swabs examined. All the isolates were identified as Mycoplasma mycoides subsp. mycoides, Small Colony type except for one isolate from pleural fluid which was identified as Mycoplasma arginini. M. mycoides subsp. mycoides, Small Colony type was isolated from samples originating from Dodoma, Iringa, Mbeya, Morogoro and Shinyanga regions where outbreaks of contagious bovine pleuropneumonia had been reported. In the examination of goats, mycoplasmas were isolated from 54 (34.0%) of the 159 examined lung samples, 41 (18.1%) of the 226 nasal swabs and 4 (40.0%) of the 10 pleural fluid samples. The species demonstrated were Mycoplasma capricolum subsp. capripneumoniae, M. mycoides subsp. mycoides, Small Colony type Mycoplasma ovipneumoniae and M. Capricolum subsp. arginini. The isolation of M. capripneumoniae in the Coast and Morogoro regions confirmed the presence of contagious caprine pleuropneumonia in the regions.     

Application of dot immunobinding on membrane filtration (MF dot) to the study of relationships within "M. mycoides cluster" and within "glucose and arginine-negative cluster" of ruminant mycoplasmas.

A total of 189 isolates from cattle, sheep and goats, allocated to two groups on biochemical grounds, have been examined by a dot immunobinding membrane-filtration (MF dot) method. Seventy glucose fermenting isolates, showing relationships with the "Mycoplasma mycoides cluster", have been compared by MF dot against polyclonal hyperimmunesera prepared against the following reference strains: M. mycoides subsp. mycoides, small colony type (SC), strain PG1 and large colony type (LC) strain Y Goat; M. capricolum strain California Kid (CK); M. species bovine serogroup 7 strain PG50, and, ovine/caprine serogroup 11 strain 2-D. The isolates fell into 5 main groups: (a) 14 serologically homogeneous isolates similar to subsp. mycoides SC PG1 (b) 4 homogeneous isolates similar to PG50 (c) 14 isolates all serologically similar to Y Goat, but heterogeneously reactive with subsp. capri PG3 and M. capricolum CK antisera (d) 7 isolates serologically similar to subsp. capri PG3, but heterogeneously reactive with subsp. mycoides SC PG1, M. capricolum CK and 2-D antisera (e) 28 isolates strongly reactive with both M. capricolum CK and serogroup 7 PG50 antisera. 119 isolates that were all glucose and arginine negative were also compared by the MF dot method with the reference strains. Most of these could be classified definitely as M. bovis (78 isolates), M. agalactiae (21 isolates) and serogroup 11 (5 isolates). 13 isolates gave a strong reaction with both M. bovigenitalium and serogroup p11 antisera. 2 isolates showed an unclassifiable pattern. The results confirm that the glucose and arginine-negative cluster strains that reacted with 2-D antiserum, also share serological relationships with the "M. mycoides cluster", albeit with a very heterogeneous pattern.     

Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capripneumoniae.

The lppA gene, encoding the lipoprotein named LppA[Mcaca] was characterised in Mycoplasma capricolum subsp. capricolum. It encodes a lipoprotein with an apparent molecular mass of 57 kDa as determined by SDS-PAGE. Using antibodies directed against recombinant LppA[Mcaca], we showed the expression of this lipoprotein in all M. capricolum subsp. capricolum by immunoblot analysis. The serum did not cross-react with other members of the Mycoplasma mycoides cluster, hence showing that LppA[Mcaca] was antigenically specific to M. capricolum subsp. capricolum. The lppA gene was conserved within the subspecies and was used for the development of a specific PCR assay for the identification of M. capricolum subsp. capricolum. The taxonomically related Mycoplasma capricolum subsp. capripneumoniae (F38) was found to contain an lppA-pseudo-gene. It showed high similarity to functional lppA genes of other mycoplasmas in the M. mycoides cluster. However, it contained interrupted open reading frames. Moreover, the nucleotide sequence of the lppA pseudo-genes in different strains of M. capricolum subsp. capripneumoniae were quite variable. Interestingly, the lppA pseudo-gene had a size similar to that of the functional lppA genes of other mycoplasmas of the M. mycoides cluster and occupied the same genomic location as the latter ones in the vicinity of the mtlD genes. This study showed that all members of the M. mycoides cluster contain each a species-, subspecies- respectively type- specific lppA gene analogue which encodes a lipoprotein that has structural and functional relationship to the surface lipoprotein LppA [MmymySC], previously named P72, of M. mycoides subsp mycoides SC, with the exception of M. capricolum subsp. capripneumoniae which seems not to express an LppA analogue.     

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.     

The in vitro effect of six antimicrobials against Mycoplasma putrefaciens, Mycoplasma mycoides subsp. mycoides LC and Mycoplasma capricolum subsp. capricolum isolated from sheep and goats in jordan

Respiratory disease in sheep and goats is a major problem in Jordan and is often associated with Mycoplasma species. Without effective vaccines, control is mainly by chemotherapy, but the uncontrolled use of antimicrobials has led to concerns about the potential development of antimicrobial resistance. The in vitro effect of chloramphenicol, florfenicol, enrofloxacin, tylosin, erythromycin and oxytetracycline was determined against 32 isolates of Mycoplasma species-M. mycoides subsp. mycoides LC (6), M. capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions of Jordan. The antimicrobial susceptibility showed some Mycoplasma species-specific differences, with M. capricolum subsp. capricolum being more susceptible to tylosin and erythromycin. Chloramphenicol and florfenicol were the least effective for all three Mycoplasma species. No trends or significant differences in antimicrobial susceptibilities were observed between sheep and goat isolates, between milk or nasal swab isolates, or between isolates from different regions of Jordan. Some isolates of M. capricolum subsp. capricolum and M. putrefaciens showed higher MIC levels with oxytetracycline, as did two isolates of M. mycoides subsp. mycoides LC with tylosin, possibly indicating signs of development of antimicrobial resistance.     

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.     

Electrophoretic analysis of isoenzymes of mycoplasma species

The purpose of this paper is to further characterize and determine relatedness among some Mycoplasma species or serogroups of significant practical veterinary interest. Twenty-four strains were examined for the presence of 35 enzymes by horizontal starch gel electrophoresis, revealing a total of 127 different electromorphs of 30 enzymes. Inter- as well as intraspecific differences were found demonstrating the application of isoenzyme studies in classification as well as epidemiology. It is concluded that the F38 group of MacOwan and group 7 of Leach constitute 2 new species, The elevation of the 2 subspecies of M. mycoides (mycoides and capri) to species level is favoured, but it is suggested that a decision be taken internationally, considering the practical consequences of a given nomenclature. Three alternative possibilities for classification are presented.Regarding identification the results suggest that the presence or absence of maltase and ornithine transcarbamylase indicate whether an isolate is related to the agent of contagious bovine pleuropneumonia or merely a representative of either the caprine LG type of M. mycoides subsp. mycoides or the classical caprine subspecies, M. mycoides subsp. capri.Key words: mycoplasmas, classification, isoenzymes, M. mycoide s-r elated groups     

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.     

First isolation of Mycoplasma capricolum subsp. capricolum, one of the causal agents of caprine contagious agalactia, on the island of Lanzarote (Spain)

During an unusually long period of bad weather, several outbreaks of caprine contagious agalactia (CCA) were reported in a number of flocks on the island of Lanzarote (Canary Islands, Spain). Clinical and subclinical mastitis in lactating goats and some cases of arthritis and pneumonia in kids were observed in the affected flocks. Mycoplasma capricolum subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with M. mycoides subsp. mycoides "large colony type" (Mmm LC) in two flocks. This is the first report of an isolation of M. capricolum subsp. capricolum on the island of Lanzarote. The finding is of epidemiological importance and could complicate plans to control the disease. The significance of this mycoplasma species in association with CCA must now be studied in detail.     

Microbiological and serological studies on caprine pneumonias in Oman

Eight of 10 typical cases of contagious caprine pleuro-pneumonia in Oman yielded strain F38-like mycoplasmas from the lungs in high titre, but no other mycoplasmas: both negative animals had been treated with tylosin shortly before death. Among 21 other lungs examined three of six cases of acute pneumonia yielded Mycoplasma ovipneumoniae; one also yielded M capricolum. M ovipneumoniae was also isolated from all eight cases of chronic pneumonia sampled from an abattoir, and from the lungs of three animals which died without overt signs of pneumonia. A single isolate of M arginini and three of unidentified mycoplasmas were also obtained from goats with and without pneumonia. Various bacterial species were isolated, none of which predominated. Antibodies to M mycoides subspecies capri (M m capri) and strain F38 were detected in sera from eight different sources. Assuming titres of 1 in 40 or more as positive in the indirect haemagglutination test used, 29 per cent of 422 serum samples had antibodies to M m capri alone, 2.6 per cent to strain F38 alone and 3.6 per cent to both organisms. These results confirm the presence of F38-like mycoplasmas in Oman, and indicate also widespread infection with M m capri. The role of the latter in caprine pneumonias in Oman requires elucidation.     

Mycoplasma mycoides subsp. capri associated with goat respiratory disease and high flock mortality

A high mortality outbreak of respiratory mycoplasmosis occurred in goats in Mexico. The clinicopathologic presentation resembled contagious caprine pleuropneumonia caused by Mycoplasma capricolum subspecies capripneumoniae. By using a battery of polymerase chain reaction assays, the mycoplasma associated with this outbreak was identified as Mycoplasma mycoides subsp. capri.     

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.     

The F38-Like Group,a New Group of Caprine Mycoplasmas?

This paper concerns the taxonomic status of the F38-like group (MacOwan), a prime determinant of contagious caprine pleuropneumonia (CCPP). Extensive biochemical and serological investigations on strain F38 are reported. Some complex serological relationships with other mycoplasma species are revealed. The results, taken in conjunction with earlier published work on geno-typic characters, lead to the conclusion that final classification of these organisms should await further comparative studies of a number of field strains with a related group of strains classified as M. capri-colum.The characterization of F38 confirms its partial relationship to the “M. mycoides group” of ovine/caprine/bovine mycoplasmas, and has also revealed a very close phenotypic relationship to the bovine mycoplasma serogroup 7, a finding of potential diagnostic and epidemiological importance.Key words: mycoplasmas, classification, F38-like group     

Effect of the caprine variant of Mycoplasma mycoides subsp mycoides on endothelium, monocytes, and complement of guinea pig, calf, sheep, and goat serum

The caprine variant of Mycoplasma mycoides subsp mycoides causes septicemia with coagulopathy in goats. Pathogenetic mechanisms that might explain the coagulopathy, the ability of the Mycoplasma to persist in the blood, and its specificity for goats were studied. Severe endothelial damage was seen by electron microscopy of goat aorta tissue exposed in vitro to 10(7) colony-forming units of mycoplasmas. The Mycoplasma did not damage 51Cr-labeled adherent cells from peripheral blood of goats. The hemolytic complement titer was reduced by 94%, 50%, 50%, and 25% in guinea pig, calf, sheep, and goat serum, respectively, 30 minutes after treatment with 8 X 10(9) colony-forming units of the Mycoplasma. Freshly prepared serum from these animal species killed the Mycoplasma. Heat-inactivated serum was not mycoplasmacidal. Complement from these 4 animal species was activated by the Mycoplasma through the classical pathway, because ethyleneglycoltetraacetic acid precipitation of serum Ca2+ inhibited activation. Proof that the classical pathway was functional in goats was not conclusive because Ca2+ supplementation of ethyleneglycoltetraacetic acid-treated serum did not restore complement activity. Endothelial damage and complement activation may explain the coagulopathy. The function that complement activation may have in the inflammatory response of this disease is not known. Difference in susceptibility of calves, sheep, and goats to M mycoides septicemia cannot be explained by species variation in complement mycoplasmacidal activity.     

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.     

Changes in lymphocyte subsets in the bronchus-associated lymphoid tissue of goats naturally infected with different Mycoplasma species

The distribution of cells containing lysozyme, S-100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus-associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3-5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1-6), M. mycoides ssp. capri (goats no. 7-12) and M. capricolum ssp. capricolum (goats no. 13-18). Microscopically, infected animals showed a moderate broncho-interstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3+ T lymphocytes, and the ratio of CD4+:CD8+ cells was > 2. The BALT showed large germinal centres mainly composed of IgG+ B lymphocytes, with numerous S-100+ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG+ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Mycoplasmas: use of polyvalent antisera for identification by indirect immunofluorescence.

It would be an advantage, under many circumstances, to be able to make use of polyvalent antisera in the process of identifying mycoplasmas. As the indirect immunofluorescence test is sufficiently sensitive and also generally accepted as being rather specific, this technique was chosen to investigate whether polyvalent antisera are applicable in routine identification of mycoplasmas. Three polyvalent sera were used, each consisting of 9 or 10 rabbit antisera raised against 29 of the more common species of the genus Mycoplasma. Twenty-six field strains were examined. One strain did not react with any of the 3 polyvalent antisera although it was later identified as M. bovigenitalium. The remaining 25 strains reacted with 1 and only 1 of the polyvalent antisera and were subsequently identified by immunofluorescence utilizing monospecific antisera. Strains of the following species were identified: M. arginini, M. bovigenitalium, M. bovis, M. bovoculi, M. canis, M. capricolum, M. cynos, M. edwardii, M. hominis, M. hyorhinis, M. molare, M. mycoides subsp. mycoides and M. opalescens. It is concluded that polyvalent antisera may be used in identification procedures and thereby permit the use of a limited number of monospecific antisera without preceding biochemical testing.     

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.