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1.
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.  相似文献   

2.
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。  相似文献   

3.
Transmissibility of Mycoplasma mycoides subsp. mycoides infection from experimentally inoculated goats to other goats and calves was studied. Eight goats and six calves were housed in an 18 m2 room. Six of the goats were inoculated endobronchially with strain D44 isolated from a natural case of polyarthritis in Ontario. These six goats died within a week of Mycoplasma septicemia. The two contact goats or the six calves never showed signs of disease and M. mycoides subsp. mycoides was not recovered from these animals. The contact goats and four calves were killed 25 days after exposure. They were all seronegative, M. mycoides subsp. mycoides was not recovered at necropsy and none had pathomorphological changes attributable to this Mycoplasma. The two remaining calves were inoculated endobronchially with 10(9) CFU of strain D44 and observed for 20 days. They never showed signs of disease and did not have significant lesions at necropsy. Both developed a significant serological response to M. mycoides subsp. mycoides, although this organism was not recovered during the experimental period or at necropsy. This study did not provide evidence for transmission of M. mycoides subsp. mycoides from endobronchially inoculated goats to contact goats or calves and endobronchially inoculated calves did not develop pneumonia. This would suggest that the infection of the goat population in Canada with this pathogen would not be a significant threat to the cattle population.  相似文献   

4.
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.  相似文献   

5.
During epidemic outbreaks in two goatherds clinical symptoms and deaths occurred in five (14%) of the 3-week-old goat kids in farm A, and in six (33%) of those in farm B. In the latter farm, three female goats aborted before the clinical symptoms in the kids emerged. Mycoplasma could be isolated from both healthy and sick goat kids and from female goats, which had diseased kids or had aborted. Three goat kids (one from herd A and two from herd B) were sent for post-mortem examination. In all these cases septicaemia caused by Mycoplasma was diagnosed. Based on the bacteriological examination the Mycoplasma strains proved to be Mycoplasma mycoides subsp. capri (Mmc). This was confirmed by the PCR examination. Mmc was isolated from several locations including from the rectum of one healthy female goat, and from two diseased kids. In addition, bacteria were detected in the small intestine in two of the necropsied kids by bacteriological and/or immunohistochemical methods. The finding suggests that Mmc may be transmitted via faeces in goatherds, kept under conventional conditions.  相似文献   

6.
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.  相似文献   

7.
Isolation and characterisation of an Indian strain of Mycoplasma mycoides subsp. mycoides LC from a case of caprine arthritis is reported in the study. The isolate was identified based on biochemical, digitonin sensitivity and growth inhibition tests. The virulence of the organism was studied by pathogenicity test in mice and goat. The antigenic and genomic profile of the isolate was compared with that of the standard strain (Y-Goat). Using different sets of primers, polymerase chain reaction was employed for rapid detection of the strain.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.  相似文献   

14.
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.  相似文献   

15.
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16.
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.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65 ℃等温条件下,60 min一步完成反应。在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色。丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应。以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL。结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法。  相似文献   

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