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.     

Disease caused by Mycoplasma mycoides subspecies mycoides LC in Hungarian goat herds

The occurrence of a goat disease caused by Mycoplasma mycoides subsp. mycoides LC in Hungary is reported. The disease occurred in two goat herds in the spring of 1999. In one herd 25% of the 4-12 weeks old kids (10 animals) while in the other herd 33% of the 6-12 weeks old kids (20 animals) became affected. The goat kids developed polyarthritis. The most severe lesions developed in the carpal joints. All animals died after 3-8 days of disease. Four dead kids were necropsied. All of them had serofibrinous and purulent polyarthritis, and in two animals bronchopneumonia, fibrinous pleuritis and meningitis were also found. In the articular exudates the presence of mycoplasmas was detected by PCR using a general mycoplasma primer. Mycoplasmas were cultured from the joints of all animals, from the abdominal parenchymal organs of two kids and from the lungs of one animal. The cultured mycoplasmas grew in strikingly large colonies, proved to be glucose positive, arginine negative and phosphatase positive, and liquefied the coagulated serum. They survived incubation at 45 degrees C for more than 24 h. Based upon their biochemical properties, the results of the immunofluorescence (IF) and growth inhibition tests and the sequence analysis of the PCR product, the cultured strains were identified as M. mycoides subsp. mycoides LC. Animals purchased in the previous autumn had been introduced to both farms. The disease may have been introduced with asymptomatic carrier animals, as earlier no similar disease had been observed at either farm.     

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.     

Genital lesions in an outbreak of caprine contagious agalactia caused by Mycoplasma agalactiae and Mycoplasma putrefaciens

This paper reports on the genital lesions observed in adult male and female goats from a commercial flock in the Extremadura region of southwestern Spain, following an outbreak of contagious agalactia syndrome caused by Mycoplasma agalactiae and M. putrefaciens. Although both species were isolated from several organs, M. putrefaciens was the only agent isolated from the genital lesions reported here, characterized by desquamative salpingitis and cystic catarrhal metritis in females and by testicular degeneration in males. Mycoplasma putrefaciens was isolated from the testes of only one of the males examined.     

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.     

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.     

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.     

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.     

Caprine mycoplasmosis by M. capricolum (author's transl)

Identified in France in 1972, this severe goat disease is now well established in goat breeding areas.Natural infection occurs as a contagious agalactia syndrome, the local lesions of which appear after a septicema stage which can lead to the death of animals in poor condition.This fatal evolution is the general rule among kids which die within a few days, after being fed with infectious milk.The experimental disease is easy to reproduce with wild strains of M. capricolum; it allows to study the immune response and also to assess the value of therapeutic agents.The strains isolated in France constitute a very homogeneous group, from a microbiological point of view; compared with reference strains, some serological variations are demonstrable, which can raise difficulties in strain identification.Microbiological diagnosis is fairly easy, but serological diagnosis is not yet standardized and a manifest cross-reaction exists with another pathogenic agent for goats, M. mycoides var. mycoides.Up to now, the epidemiological characteristics of this mycoplasmosis are to a great extent unknown; recovered animals are carriers which allows the disease to be maintained in flocks, but the features of this carrier state (frequency, duration, predisposing factors) are not well known. Screening of such goats remains uncertain.Specific treatment must be carried out with some antibiotics; indications and results are to be discussed.     

Isolation of Mycoplasma bovis from bovine clinical mastitis cases in Northern Greece

Mycoplasma bovis was detected in 18/219 (8.2%) quarter milk samples collected from cases of bovine clinical mastitis in Northern Greece between November 1997 and March 1999. The cases occurred in 2/37 (5.4%) of the herds examined. The micro-organism was isolated from bulk milk samples (BTS) from the two positive herds but was not isolated from 111 composite milk samples collected from clinically healthy cows from all 37 herds. Isolates were identified as M. bovis by polymerase chain reaction (PCR) assay. Other micro-organisms were also isolated from the M. bovis positive samples. The M. bovis-positive cows had all been imported into Greece from other European countries.     

Indirect infection of cattle with contagious bovine pleuropneumonia.

Hay infected with Mycoplasma mycoides sub-species mycoides (M mycoides) was fed to six animals on three occasions. Five animals developed complement-fixing antibody in their sera and gave a positive reaction to the comparative intradermal allergic test. No animal died from contagious bovine pleuropneumonia (CBPP) but when they were killed, three had unequivocal lesions of the disease. M mycoides was isolated from the animals with these lesions but not from the others. CBPP was transmitted to two animals put in contact with those animals showing disease, and M mycoides was recovered from lesions in both animals. It is suggested that when unexplained outbreaks of CBPP occur, the possibility of indirect transmission should be considered.     

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.     

Prevalence of contagious mastitis pathogens in bulk tank milk in Prince Edward Island

The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.     

Incidence of Mycobacterium paratuberculosis in raw sheep and goats' milk in England, Wales and Northern Ireland

A blind survey of 104 raw sheep and goats' milk samples (90 goat, 14 sheeps) from bulk tanks on farms throughout England, Wales and Northern Ireland was carried out over a 5-month period (January-May 1998) in order to determine the incidence of Mycobacterium paratuberculosis. Each milk sample (100 ml) was divided into two 50ml portions. One portion was decontaminated with 0.75% hexadecylpyridinium chloride for 5h before culture on slopes of Herrold's egg yolk medium and in BACTEC radiometric medium. The second portion was subjected to immunomagnetic separation followed by IS900 PCR (IMS-PCR). The IMS-PCR assay was employed in order to provide a more rapid indication of the presence of M. paratuberculosis in each milk sample than is possible by culture. Information on the Johne's disease status of the sheep and goat herds that took part in the survey was not sought at the time of milk sampling. However, it subsequently emerged that at least some of the herds whose bulk milk was tested during this study were previously or currently infected with Johne's disease. Overall, during this survey one raw goats' milk sample tested positive for the presence of M. paratuberculosis by IMS-PCR (<1% of milk samples tested) but no viable M. paratuberculosis were isolated by culture. The results of this study suggest that bulk raw sheep and goats' milk from these regions of the UK may not represent significant vehicles of transmission of M. paratuberculosis to humans.     

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.     

Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of bovine Johne's disease (BJD) in lactating Indian dairy cattle

Present study is the first attempt to evaluate an indigenous milk ELISA with milk culture, standardize milk PCR, estimate lacto-prevalence of Map and genotype Map DNA from milk samples in few Indian dairy herds. In all 115 cows were sampled from 669 lactating cows in six dairy herds from three districts of North India. Fifty milk samples (four herds) were screened by three tests (milk culture, m-ELISA and m-PCR). Lacto-prevalence of Map in four dairy herds was 84.0% (50.0% in fat and 62.0% in sediment). Screening of both fat and sediment increased the sensitivity of culture. Colonies appeared between 45 and 120 DPI. In indigenous m-ELISA, protoplasmic antigen derived from native Map 'Bison type' strain of goat origin was used. Screening of 115 lactating cows by m-ELISA ('herd screening test') detected 32.1% positive lactating cows (lacto-prevalence). Sensitivity of ELISA was 28.5% and 42.8% in single point cutoff and S/P ratio, respectively. Lacto-prevalence of JD was high in dairy herds (66.6-100.0% by culture and 20.0-50.0% by m-ELISA). DDD farm, Mathura had very high (95.8%) and moderate prevalence of Map and lacto-antibodies, respectively. All cows were clinically suffering from JD. Specific IS 900 PCR was standardized in decontaminated fat and sediment of milk samples. DNA isolated from decontaminated pellets was amplified and characteristic 229 bp band was confirmatory for Map. Of the 50 milk samples, 6.0% were positive in m-PCR. The test needs further standardization. Map DNA were genotyped as Map 'Bison type' by IS 1311 PCR-REA. Of the three tests, milk culture was most sensitive followed by m-ELISA and m-PCR. Map DNA isolated from milk samples of dairy cattle were first time genotyped as Map, 'Bison type' in India. High prevalence of Map in milk of dairy herds, posed major health hazard for calves and human beings.     

Experimental infections of goats with Mycoplasma mycoides subspecies mycoides, LC type

In five experiments 29 goats were infected experimentally by five different routes with a strain of Mycoplasma mycoides subspecies mycoides, LC type, isolated from a contagious caprine pleuropneumonia-like outbreak on a farm in northern Sweden. All the goats were colonised except those inoculated subcutaneously with small doses. In its pattern of pathogenicity this strain was similar to other experimentally tested strains except that peroral infection in kids produced no clinical signs. A 'contact' goat was also colonised but the clinical signs seen in it were probably due to a concomitant infection with Pasteurella haemolytica.     

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 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.     

Mastitis caused by Mycoplasma mycoides subspecies mycoides (large colony type) in goat flocks in Spain

We describe three different outbreaks of mastitis caused by M. mycoides subspecies mycoides LC type (Mmm LC) in three goat flocks from the Extremadura Region of south-west Spain. Thirty-two fast-growing isolates were obtained on Hayflick's and Friis's media with inhibitors from different specimens. All were identified as Mmm LC in spite of their cultural, biochemical and serological features.