【作者】 储岳峰；

【导师】 柳纪省；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 博士

【摘要】 山羊(接触)传染性胸膜肺炎(Contagious caprine pleuropneumonia, CCPP)是由山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp. capripneumoniae, Mccp)引起的山羊呼吸道传染病,是OIE法定报告动物传染病之一。我国于2007年分离出1株Mccp,并在同年证明上世纪50年代分离的3株“山羊传染性胸膜肺炎”病原也应归类于Mccp,证实了CCPP在我国存在。随着CCPP的发现,弄清其在我国现阶段的流行状况及其起源、研制安全有效的疫苗等成为亟需解决的问题。本研究的内容和结果如下:1.对我国已报道与CCPP相关的3种支原体Mccp、绵羊肺炎支原体(Movi)和丝状支原体山羊亚种(Mmc)的培养和形态特征进行了观察,发现Mccp、Movi生长缓慢,液体培养物不浑浊或轻微浑浊,Mccp菌落直径200~500μm,有中心脐,Movi直径70~350μm,无中心脐;Mmc生长速度快,液体培养物浑浊,菌落直径300~1800μm,有中心脐;根据培养表现和菌落形态可对这3种支原体进行初判。免疫印迹试验发现Mccp与Mmc之间存在双向交叉反应,Mmc血清与Movi之间、Movi血清与Marg之间存在单向交叉反应,表明以多抗血清或全菌抗原为基础建立CCPP诊断方法不可靠。2.通过PCR检测和病原分离鉴定,从13个省市(区)送检的122份疑似CCPP的病料中检出Mccp、Movi、Mmc、山羊支原体山羊亚种(Mcc)和精氨酸支原体(Marg)共5种支原体。23.8%的Mccp检出率和6株Mccp的分离,证实CCPP在我国现阶段流行;但Movi的高检出率(58.2%)和分离率(52.4%)是否具有重要的致病意义尚需进一步研究;而Mmc的低检出率(1.64%)和2株Mmc的分离提示Mmc对我国养羊业的影响需进一步证实;国内首次分离到1株Mcc表明羊支原体病病原日趋复杂;而Mccp与溶血性曼氏杆菌(Mh)、多杀性巴氏杆菌(Pm)、隐秘化脓杆菌(Ap)、山羊痘病毒(GTPV)、羊口疮病毒(ORFV)等多病原混合感染给羊病的防制带来了新的挑战。3.用Mccp特异性间接血凝试验,对11省市(区)的2281份山羊和绵羊血清进行了血清学调查,结果10省市(区)呈现不同程度CCPP阳性,平均阳性率16.40%,从血清学角度证实CCPP在我国现阶段流行。而绵羊血清中Mccp抗体阳性(14.77%,n=1510)在CCPP流行和危害中的意义需进一步确定。4.对我国6株Mccp的H2基因序列进行分析,发现我国流行的Mccp属于2种不同的基因型:A和Ad,且当前流行菌株与上世纪50年代流行菌株具有遗传相似性,表明CCPP在我国长期存在。5.国内外首次完成了1株Mccp(M1601株)的全基因组测序,其基因组全长1018102 bp,G+C含量为23.97%,含961个编码序列。6.国内首次完成了CCPP灭活疫苗(M1601株)的实验室研制。将M1601株菌体悬液用甲醛溶液灭活后与佐剂603M乳化制成疫苗,每毫升疫苗中含0.125 mg M1601菌体蛋白。无论羊只大小,颈部皮下注射3 ml,可使免疫羊获得4/5或以上免疫保护,免疫持续期至少6个月,且对不同生理状况的山羊均安全。疫苗在2~8℃可保存15个月。7.我国“山羊传染性胸膜肺炎”与英文专业名词“CCPP”概念不一致,建议将由Mccp引起的山羊疾病改称“山羊接触传染性胸膜肺炎”,与CCPP统一;而将其它支原体引起的山羊和/或绵羊肺炎称之为“羊支原体性肺炎(Mycoplasmal pneumonia of sheep and goats, MPSG)”。更多还原

【Abstract】 Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) is a disease of goats which causes high morbidity and mortality. It is one of the listed diseases by the Office International des Epizootes (OIE). Mccp was isolated from Chinese goats in 2007. But in the same year, three strains of pathogens from CCPP cases occurred in 1950s were also reclassified as Mccp, which indicated that the true CCPP caused by Mccp might be present in China for a long time. These findings prompted us to pay more attention on CCPP and its causative agents. It is therefore indispensable and imperative to unravel the epidemiological status in current China and to develop vaccine against Mccp. This study focused on the epidemiological investigation of Mccp and the other mycoplasmas reported to be related with CCPP in China, and development of an inactivated vaccine for CCPP.It can provide the important materials for CCPP research and contribute to the prevention and control of CCPP in China. The main contents of research included as below:1. Comparative observation on some biological characteristics of Mccp, Mycoplasma ovipneumoniae (Movi)and Mycopalsam mycoides subsp. capri (Mmc): (1) Characteristics of cultivation and colonial morphology of Mccp, Movi and Mmc, which all reported in China, were studied using type strain and several field strains. Mccp and Movi grew slowly and the broth cultures were both clear; Mccp colonies on the solid medium had typical“fried-egg”morphology with the diameter of 200~500μm, but Movi colonies had centerless morphology with the diameter of 70~350μm. On the other hand, Mmc grew rapidly and could make the liquid medium turbid. The Mmc colonies on the solid medium had typical“fried-egg”morphology and the diameter could reach 300~1800μm. So the growth speed in the medium and colonial morphologies of the three mycoplasma species were obviously different, which contributed to early differentiate the culture of mycoplasma from the clinical materials. (2) The SDS-PAGE patterns of 8 mycoplasma species related with pulmonary disease of goats were comparatively observed using type strain and several isolates, including Mccp, Movi, Mmc, Mycoplasma capricolum subsp. capricolum (Mcc), Mycopalsam mycoides subsp. mycoides Small Colony (MmmSC), Mycopalsam mycoides subsp. mycoides Large Colony (MmmLC), Mycoplasma arginini (Marg) and Mycoplasma agalactiae (Maga). The results showed that these mycoplsma species were divided into 5 different groups when analyzed by the software NTsys-2.10e with the similarity coefficient of 75%: Mccp/Mcc group, MmmLC/Mmc/MmmLC group, Maga group, Movi group and Marg group. The immunogenic proteins of the 8 mycoplamsa species which reacted with antiserum against Mccp strain M1601, Mmc strain PG3 and Movi strain Y98 were then detected by using the Western-blot method, respectively. It was found that the two-way corss reaction existed between Mccp and Mmc, one-way cross reaction existed between Mmc sera and Movi, and between Movi sera and Marg. The results indicated that it was insufficient to develop the methods to accurately diagnose CCPP based on the polycolonal antibodies or antigens prepared from the full mycoplasmal proteins.2. An etiological investigation of CCPP in China—molecular detection, isolation and characterizaton of causative agents form the suspected CCPP cases: Five mycoplasma species including Mccp, Movi, Mmc, Mcc and Marg were detected by PCR and/or isolated by cultivation from 122 clinical materials collected from 13 provinces (autonomous regions/cities) in China. (1) The detection rate of Mccp was 23.8% (n=122) and 6 strains of Mccp were successfully isolated, Mccp was also detected and clone purified from 2 old Chinese strains, C87001 and C87002, which isolated form 1950s. The findings confirmed the prevalence of CCPP in China. (2) The detection rate and isolation rate of Movi were 58.2% (n=122) and 52.4% (n=63), respectively. But the pathogenesis significance of the high detection rate and isolation rate of Movi should be further confirmed, because Movi is one of the common floras located in the respiratory tract of goats, also because of the high heterogeneity among Movi strains. (3) Only 2 strains of Mmc were isolated and 1.64% (n=122) clinical materials were positive to Mmc detected by PCR. It prompted that the real impact of Mmc on the Chinese goat industry should be further investigated, especially when the causative agents of CCPP-related diseases were nowadays referred to Mmc in many reports. (4) A strain of Mcc was isolated for the first time form goat in China and identified by PCR and H2 gene DNA sequence analysis. So the prevalence of Mcc is also alarming in this country. (5) Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Arcanobacterium pyogenes (Ap), Goatpox virus (GTPV) and Orf virus (ORFV) were also synchronously detected from the suspected CCPP cases. These findings challenge the existing plans for the prevention and control of goat diseases in China.3. Sero-prevalence of CCPP in China: A total of 2281 sera from goats and sheep collected from 11 provinces (autonomous regions/cities) in China were detected for Mccp antibodies with an indirect hemagglutination test (IHAT), which established by employing sheep erythrocytes sensitized with secreted polysaccharide extracted from Mccp strain M1601 culture supernatant. 374 (16.40%) sera from 10 out of 11 provinces were positive to Mccp. Moreover, the positive rate was 14.77% among sheep sera, which indicated Mccp might be present in sheep flocks. But the epidemiological significance of this finding in China is still unclear because no test was performed to detect or isolate Mccp from sheep up to now.4. Molecular epidemiology on CCPP in China: The H2 locus of 6 strains of Mccp isolated from China were cloned, sequenced and analyzed by the method described by Lorenzon et al. (2002). Six strains of Mccp were divided into 2 different genotypes—genotype A and genotype Ad. The isolates from 2007~2010 were genetically homology with the 2 strains from 1950s, which indicated CCPP should be endemic in China for a long time.5. Complete genome sequence of the Mccp strain M1601: Whole-genome sequencing of Mccp was completed for the first time. It was performed on the Illumina GAIIx platform and the reads were assembled using the Abyss software. The genome of M1601 consists of a single chromosome comprising 1,018,102 bp. The chromosome has relatively low GC content (23.97%). There are 961 open reading frames (ORFs) and 37 non-coding RNA genes in the genome. The coding density of the genome is 77.84%. The results contributed to understand the pathogenesis of Mccp and to develop new technologies for control of CCPP at a genomic level. 6. Development of an inactivated vaccine for CCPP: The M1601 strain of Mccp grown in the Thiaucourt’s broth medium was harvested by centrifugation; the pellet was washed 2 times and re-suspended in sterile normal saline. After the protein content was measured by the bicinchoninic acid (BCA) method by comparison with a bovine albumin standard, the suspension was diluted to 0.25 mg/ml using sterile normal saline contained 0.2% formalin for inactivation. The inactivated suspension was then mixed into a new nanometer adjuvant named 603M with a volume ratio of 1︰1. Three batches of Mccp bacterin were produced in the laboratory and were then used to evaluate the safety and protection efficacy to the goats. The results showed that all the goats including pregnant goats, inoculated subcutaneously with 3 ml bacterin, were safe and no adverse side effects were found. The protection rate of each batch of bacterin was more than 4/5 and duration of immunity was at least 6 months. If stored at 2~8℃, the vaccine had a shell life of 15 months.7. Proposal of re-classification and designation of the CCPP-related disease in Chinese: Only the disease of goats caused by Mccp can be referred to CCPP. The pulmonary diseases of goats and sheep caused by other mycoplasmas species such as Mmc/MmmLC, Mcc and Movi should be referred to Mycoplasmal pneumonia of sheep and goats (MPSG).更多还原