一株山羊支原体山羊肺炎亚种的分离鉴定与分子特征

从送检的山羊肺炎肺脏中成功分离到一株支原体，经过3次克隆纯化后进行生化试验、电镜观察、PCR及酶切、基因特征鉴定，结果显示分离物SD3属于山羊支原体山羊肺炎亚种成员。将培养物经气管接种2只山羊可引起1只山羊典型发病，体温升高至41．5℃，IgG和IgM抗体效价明显升高，其中IgG抗体变化与临床表现基本同步。剖检发现肺脏发生严重病变，并从中再次分离到该病原体。     

山羊支原体的分离与鉴定

1997年对怀化市山羊疫病血清学调查时发现山羊传染性胸膜肺炎阳性率为7.25%。为证实本病的存在，从1998年以来共采集山羊肺脏病理材料60份，分离出二株支原体，将分离株接种山羊气管后复制出本病。根据国际兽疫局的标准诊断方法以及分离物培养特性、显微镜观察、生长抑制试验、生化试验等，初步鉴定为丝状支原体山羊亚种和绵羊肺炎支原体。     

山羊支原体山羊肺炎亚种湖北株的鉴定及其外膜蛋白的免疫原性研究

从湖北某地山羊肺脏中分离支原体,经多次克隆纯化后进行生化试验、显微镜观察、PCR及酶切分析、基因特征鉴定以及用动物致病性试验对该菌株进行了鉴定,命名为GH2,进一步通过免疫印迹对GH2株外膜蛋白及GH2株全菌蛋白免疫原性进行分析,并用抗Y98血清和抗PG3血清进行比较,筛选GH2株特异性的外膜蛋白,结果表明GH2株为山羊支原体山羊肺炎亚种,存在于GH2外膜蛋白中的相对分子质量大约为60和49.7 ku的蛋白可能是其主要的免疫原性蛋白,也是GH2株区别于丝状支原体山羊亚种标准株PG3和绵羊肺炎支原体标准株的主要特异性抗原.这一结果为山羊支原体山羊肺炎亚种的诊断和疫苗研制提供了良好的理论基础.     

广西山羊传染性胸膜肺炎病原的二重PCR快速诊断方法的建立

根据绵羊肺炎支原体标准株Y98和丝状支原体山羊亚种标准株PG3的16S rRNA两端的保守区序列设计了2对引物,建立了广西山羊传染性胸膜肺炎病原的二重PCR快速检测方法.试验结果显示,所建立的二重PCR能特异地扩增绵羊肺炎支原体和丝状支原体山羊亚种的基因片段,其敏感性可达lPg,,用建立的二重PCR检测了20份临床病例肺组织,检出率为70%(14/20),其中6份扩增出丝状支原体山羊亚种的基因片段,10份扩增出绵羊肺炎支原体的基因片段,有2份同时扩增出两种支原体的基因片段.培养法检出率为40%(8/20),而这8份病料均为PCR阳性.     

山羊传染性胸膜肺炎病原分离与鉴定

本试验从传染性胸膜肺炎的山羊体内分离获得两株支原体Y1和Y2，通过病原分离、形态学观察、生化试验、HA、生长抑制试验和代谢抑制试验等试验，证实Y1和Y2的形态与培养特性、生化反应特性和血清学特性分别与模式株丝状支原体山羊亚种PG3和绵羊支原体Y98相接近；动物回归试验成功复制出山羊传染性胸膜肺炎的典型临床症状和病理剖解变化。结果表明Y1和Y2分别为丝状支原体山羊亚种和绵羊支原体。     

贵州山羊传染性胸膜肺炎病原的分离鉴定

经临床症状，病理剖检，病原分离培养，生化试验，生长抑制试验，代谢抑制试验，人工接种发病，间接血凝诊断，对贵州省习水县和罗甸县山羊群以呼吸道疾患为主要特征的传染病进行诊断，确诊为山羊传染性胸膜肺炎，分离出的两株支原体均为丝状支原体山羊亚种PG3，分别定名为贵习PG3／99和贵罗PG3／99。     

山羊传染性胸膜肺炎研究进展

山羊传染性胸膜肺炎是由山羊支原体山羊肺炎亚种引起的一种山羊常见的高度接触性呼吸道传染病,有传染快速,发病率、死亡率高的特点,给山羊养殖业带来巨大损失。本文从病原学、流行病学、临床症状以及诊断与防控方面对山羊传染性胸膜肺炎进行综述,帮助读者更加全面、深入的了解本病,以期为山羊传染性胸膜肺炎的诊断和防治提供新思路。     

山羊支原体山羊肺炎亚种培养基的筛选及生长曲线测定

为筛选出山羊支原体山羊肺炎亚种的适宜培养基,将山羊支原体山羊肺炎亚种分离株M1601分别接种Thiaucourt肉汤、MEM-KM2和TSA 3种不同的培养基,测定其在3种不同培养基中生长滴度、生长速度,并测定了M1601在最适培养基中在不同培养阶段的生长滴度。结果表明,添加2g/L丙酮酸钠和150mL/L马血清的Thiaucourt肉汤培养基最适宜山羊支原体山羊肺炎亚种的生长,生长滴度可达109 CCU/mL,在培养6h后开始进入对数生长期,培养60h后进入稳定期,培养72h时后进入衰亡期。此试验结果为山羊支原体山羊肺炎亚种培养特性研究提供了参考数据。     

山羊传染性胸膜肺炎病原的分离鉴定

从内蒙古地区山羊病料中分离出疑似山羊传染性胸膜肺炎的病原,经染色镜检、生化鉴定、血清学等方法初步鉴定为山羊支原体山羊肺炎亚种.参照已知山羊支原体ADI基因设计的引物进行PCR扩增,通过电泳分析得到与文献报道相一致的大小为316 bp的条带,最终确定为山羊支原体山羊肺炎亚种.     

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.     

Humoral immune responses following experimental infection of goats with Mycoplasma capricolum subsp. capripneumoniae.

Goats housed in microbiologically secure facilities were experimentally endobronchially infected with Mycoplasma capricolum subsp. capripneumoniae (Mccp), causal agent of contagious caprine pleuropneumonia (CCPP). The animals were monitored over an 8-week period post-infection (p.i.). Elevated temperatures were observed 2-7 days p.i., reaching a maximum of 41.5 degrees C in one animal (1884). By 8 weeks p.i. the infection was successfully cleared, with no Mccp being recovered from the lungs, serum or nasal passages. Mccp was not isolated from serum throughout the experiment, either directly by culture or indirectly via polymerase chain reaction (PCR). Humoral immune responses against Mccp capsular polysaccharide (CPS) were generally poor when measured by ELISA. CPS antigen was present in the serum of all infected animals early in the infection (day 14 p.i.), although in one animal (1855) CPS antigen persisted throughout. This was the only animal to exhibit a serious cough (day 5-19 p.i.). Successful diagnosis of CCPP was achieved using two different types of latex agglutination test (CPS antibody and CPS antigen detection test), immunoblotting and a blocking ELISA, although the latter lacked sensitivity until later in the infection (35-40 days p.i.). Only a single animal (1855) was detected positive using the current complement fixation test (CFT). Strong immune responses to protein antigens were detected by IgG and IgM immunoblotting from the first time point at day 14 p.i. IgM immunodominant bands of 220, 85, 62 and 40kDa were observed in the 3 infected animals and from CFT-positive CCPP field sera. Band intensity gradually diminished throughout the experiment. IgG immunodominant bands of 108, 70, 62, 44, 40 and 23kDa were shared between experimentally-infected and field sera, with band intensity either remaining unchanged or increasing from day 14 p.i. These bands were not present using pre-infection sera. Of the diagnostic tests used, only the CPS antibody detection latex agglutination test and IgG immunoblotting gave positive diagnoses throughout the entire period post-infection (days 14-53 p.i.).     

Antibody response to Mycoplasma capricolum subsp. capripneumoniae bacterium in small holder dairy goats in Tanzania

Contagious caprine pleuropneumonia (CCPP), an Office International des Epizooties listed disease, can cause significant levels of morbidity and mortality in goats in Africa, the Middle East, and Western Asia. A cross-sectional study was conducted to determine the seroprevalence of CCPP, in two contrasting administrative districts of northern Tanzania, namely, Babati and Arumeru. A total of 337 serum samples were collected from January to July 2010, from apparently clinically healthy unvaccinated dairy goats breeds of different ages and sexes. Samples were subjected to monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the specific measurement of antibodies to Mycoplasma capricolum subsp. capripneumoniae bacterium. The overall animal flock and village-level seroprevalence of CCPP was found to be 3.3 % [95 % confidence interval (CI)?=?1.6–5.8], 9.6 % (95 % CI?=?4.7–16.9), and 31.5 % (95 % CI?=?15.6–56.5), respectively. There was no evidence (p?=?0.659) of differences in seroprevalence between samples from the two administrative locations. Despite the fact that there was no significant difference (p?>?0.05) in seropositivity between sex and age and between breeds sampled, there was a significant difference (p?<?0.05) in seropositivity between the different physiological status groups examined with the non-lactating (dry) category of goats showing a higher seroprevalence. The findings of this survey revealed evidence of dairy goat exposure to M. capricolum subsp. capripneumoniae, although at a low prevalence. It is therefore advisable to include CCPP serology in the seromonitoring program so as to give a better indication of flock immunity. This should lead to the establishment of appropriate CCPP control measures in smallholder dairy goat flocks, which are increasingly being recognized for their value as a vital source of livelihood for resource poor livestock keepers in Tanzania.     

山羊支原体山羊肺炎亚种单克隆抗体的制备及抗体结合抗原表位的筛选

本研究旨在制备山羊支原体山羊肺炎亚种(M.capricolum subsp.capripneumoniae, Mccp)的单克隆抗体并筛选与抗体结合的抗原表位。试验用甲醛灭活的Mccp免疫BALB/c小鼠,运用传统的细胞融合技术进行融合获得杂交瘤细胞,亚克隆,制备单克隆抗体腹水,采用酶联免疫技术（ELISA）和免疫印迹（Western blotting）技术鉴定单克隆抗体的特异性及胶内酶切鉴定与抗体结合的抗原表位。最终成功筛选获得5株单克隆抗体,鉴定到3个与抗体结合的抗原表位,为下一步科学研究提供了初步且可靠的试验基础。     

山羊支原体山羊肺炎亚种和多杀性巴氏杆菌双重 PCR 检测方法的建立

为建立一种能够同时检测山羊支原体山羊肺炎亚种和多杀性巴氏杆菌的双重 PCR 方法,本研究采用2对特异性引物,对退火温度和引物浓度比进行了优化,成功建立了一种能同时检测上述两种病原的双重 PCR 方法。结果显示,该方法具有很好的特异性,仅对山羊支原体山羊肺炎亚种和多杀性巴氏杆菌有扩增,而对其他常见的羊呼吸道病原无扩增;该方法对两种病原的检测限分别为32 pg 和50 pg,与单独 PCR相同;26份临床样品中山羊支原体山羊肺炎亚种的检出率为23．1％,多杀性巴氏杆菌的检出率为26．9％。所建立的双重 PCR 具有特异性好、灵敏度高的特点,为临床上这两种病原感染的快速诊断和流行病学调查等提供了更为有用的手段。     

The pathogenicity and pathogenesis of Mycoplasma capricolum subsp. capripneumoniae (F38) in the caprine mammary gland

The right mammary gland of 12 lactating goats was inoculated intracisternally with 1 ml of Mycoplasma capricolum subsp. capripneumoniae (Mcc) containing 10>⁶ colony-forming units (CFU), while their left mammary halves received 1 ml of sterile PPLO broth only. Two goats served as uninfected controls. The clinical mastitis that developed in the infected mammary halves within 24 h was initially acute but became increasingly chronic by the end of the experiment at 24 days post inoculation (DPI). The disease was characterized by atrophy of the infected mammary halves, leading to marked agalactia and an increase in somatic cell counts, with a preponderance of neutrophils initially and lymphocytes later. The Mycoplasma was re-isolated from infected mammary secretions up to 16 DPI but not from blood. Histopathology revealed that the mastitis was acute and purulent initially, followed by infiltration of lymphonuclear cells and fibroplasia in the lymphomononuclear cells and fibroplasia in the interacinar tissue, and later by massive fibrosis. Immunohistology demonstrated the presence of Mycoplasma-like bodies localized mainly on the surface of acinar/duct epithelial cells. The studies showed that Mcc was highly pathogenic in the caprine mammary gland.     

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.     

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.     

Genetic evolution of Mycoplasma capricolum subsp. capripneumoniae strains and molecular epidemiology of contagious caprine pleuropneumonia by sequencing of locus H2

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in developing countries. Its exact distribution is not well known, despite the fact that new diagnostic tools such as PCR and competitive ELISA are now available. The authors developed a study of the molecular epidemiology of the disease, based on the amplification of a 2400 bp long fragment containing two duplicated gene coding for a putative membrane protein. The sequence of this fragment, obtained on 19 Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from various geographical locations, gave 11 polymorphic positions. The three mutations found on gene H2prim were silent and did not appear to induce any amino acid modifications in the putative translated protein. The second gene may be a pseudogene not translated in vivo, as it bore a deletion of the ATG codon found in the other members of the "Mycoplasma mycoides cluster" and as the six mutations evidenced in the Mccp strains would induce modifications in the translated amino acids. In addition, an Mccp strain isolated in the United Arab Emirates showed a deletion of the whole pseudogene, a further indication that this gene is not compulsory for mycoplasma growth. Four lineages were defined, based on the nucleotide sequence. These correlated relatively well with the geographical origin of the strains: North, Central or East Africa. The strain of Turkish origin had a sequence similar to that found in North African strains, while strains isolated in Oman had sequences similar to those of North or East African strains. The latter is possibly due to the regular import of goats of various origins. Similar molecular epidemiology tools have been developed by sequencing the two operons of the 16S rRNA gene or by AFLP. All these various techniques give complementary results. One (16S rRNA) offers the likelihood of a finer identification of strains circulating in a region, another (H2) of determining the geographical origin of the strains. These tools can make a very useful contribution to understanding the epidemiology of CCPP.