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.     

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.     

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.     

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.     

Classification and identification of ovine and caprine Mycoplasmas.

The purpose of this investigation was to give a survey of the classification of ovine/caprine mycoplasmas as a basis for the identification of strains isolated from sheep and goats. A total of 13 strains representing 13 species and/or serogroups were biochemically examined, and serological cross-titrations were performed using metabolism inhibition, growth inhibition and immunofluorescence. Serogroup 6 (Al-Aubaidi) was found to be identical with Mycoplasma capricolum.The results of identification of 57 isolates, sent to the reference centre from different countries, are given.On the basis of the above investigations and a comparison of some of the classification systems described in the literature, it is concluded that the following species have been isolated from goats and/or sheep: M. agalactiae, M. arginini, M. bovis, M. capricolum, M. conjunctivae, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. ovipneumoniae, M. putrefaciens, Acholeplasma granularum, A. laidlawii and A. oculi. In addition, both Ureaplasmas and strains representing 6 serogroups (groups 5, 7, 10 and 11 of Al-Aubaidi and groups 17 and 18 of Cottew) have been isolated. These serogroups ought to be finally species-classified as soon as possible. kw|Keywords|k]ovine/caprine mycoplasmas; k]classification; k]identification     

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.     

Inactivation of Mycoplasma species involved in contagious agalactia

The suitability of 5 agents for the inactivation of different field strains of the four mycoplasma species associated with contagious agalactia syndrome in goats, i.e. Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens, was investigated. Immunoprophylaxis of this syndrome is still based on inactivated vaccines, which traditionally use formalin as the inactivating agent. Moreover, the limited information existing about this type of vaccine is only based on assays against Mycoplasma agalactiae. Our results showed that formalin (0.1%, 37 degrees C during 16 hours) and phenol (0.5%, 24 hours) were effective against all species tested. Surprisingly, binary ethileneimine (BEI), a classical virus-inactivating agent, also proved to be very effective when it was used in a 0.1 M concentration over 24 hours. With heat treatment, every species was inactivated at 60 degrees C. No satisfying results were obtained with purified saponin. To evaluate the harmful effects of each agent on mycoplasmal proteins, a representative strain was subjected to an effective inactivation protocol with each agent, which was monitored by Western immunoblotting. Immunoblotting was performed using sera of animals inoculated with the respective mycoplasma species, to compare the effect of all the agents on treated strains with untreated strains. The results confirmed that phenol, BEI and to a lesser extent also formalin inactivated all species without causing a significant damage while heat caused stronger damage on surface proteins. Future in vivo studies should be conducted because, as recently shown, the combined use of a suitable inactivant and adjuvant could give rise to the induction of certain cytokines and strong antibody production of a specific isotype pattern, thus opening ways to develop more efficacious inactivated vaccines against contagious agalactia.     

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.     

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.     

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.     

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.     

Studies of the origin of false positive reactions in the serodiagnosis of contagious bovine pleuropneumonia

Four groups of cattle were experimentally immunised by four mycoplasma species of "mycoides-like" group, Mycoplasma (M) capricolum, M. mycoides subsp. mycoides (LC), M. mycoides subsp. capri and M. species group 7 of LEACH (PG50). They were then bled weekly during 2 months to establish antibodies kinetics against homologous and heterologous antigens. The standard method of complement fixation test (CFT) used in Europe and a new ELISA test for diagnosis of contagious bovine pleuropneumonia were performed in comparison with passive haemagglutination test (PHA) against antigens used for experimental immunisation. Cross reactions obtained are rather equal to the degree of similitude between these mycoplasma species. With CFT-cross reactions are transitory and occur only while homologous titers are very high, particularly with "PG50" and the two caprine mycoides strains. ELISA results using a threshold of positivity of optical density of 0.20, were similar to that obtained with CFT except ELISA specificity is not so different from CFT one. This experimental model could explain some natural situations.     

Microbiological survey for Mycoplasma spp. in a contagious agalactia endemic area

In this work, we report a microbiological survey for Mycoplasma spp. undertaken between 2001 and 2002 in 28 goat herds in Gran Canaria, Spain, an area where contagious agalactia is endemic. All herds were randomly selected and represented approximately 15.5% of the total goat population of the island. A variable number of milk, articular and auricular swab samples were collected from each flock and cultured in specific mycoplasma culture media. There was a total of 38.5% positive flocks from which 37 mycoplasma isolates were obtained. In contrast with previous data obtained in Spain, our results showed that the large colony variant of M. mycoides subsp. mycoides (Mmm LC) was the most commonly isolated agent associated with contagious agalactia. This species was isolated from 90% of the positive herds and accounted for 54.1% of all isolations. M. agalactiae was isolated from 40% of the positive herds (27% of all isolations) and in six herds M. arginini was isolated (18.7% of all isolations). No M. capricolum or M. putrefaciens strains were isolated. Mycoplasmas were isolated from 21 milk samples, 15 ear canals swabs and one articular sample. The association of several species was reported in several herds. These results are at variance with previous serological studies, which indicated a higher disease prevalence, and suggest that it could be necessary to use detection techniques such PCR to confirm the existence of contagious agalactia in goats.     

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.     

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.     

Detection of antibodies to mycoplasmas in South American camelids

Indirect haemagglutination tests on sera from 757 South American camelids (alpacas, llamas and vicunas) carried out in the Andean region of Peru, revealed evidence of exposure mainly to Mycoplasma mycoides subspecies mycoides LC. The incidence of detectable antibodies to this mycoplasma in 554 alpacas was 5.0 per cent and in 141 llamas 15.6 per cent. Antibody to Mycoplasma capricolum and the F38 biotype was detected in 0.9 per cent and 0.2 per cent of alpacas, respectively. In a group of 62 vicunas only one reactor to both M m mycoides LC and M capricolum was observed. No reactors to M mycoides subspecies capri or M agalactiae were observed in the flocks examined. Antibodies to mycoplasma were also detected in nine out of 10 goat flocks tested. The incidence of antibodies to M m mycoides LC was 13.8 per cent, 3.8 per cent for M capricolum and 1.8 per cent for the F38 biotype. In a group of 110 sheep, six reactors (5.5 per cent) to M m mycoides LC and one (0.9 per cent) to F38 were observed. The implications of these results are discussed in relation to the involvement of mycoplasmas in existing disease in camelids in Peru.     

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.