Biochemical Characterization of Mycoplasma bovirhinis,Mycoplasma dispar and Recent Bovine Isolates of Mycoplasma canis

The pattern and kinetics of substrate utilization by the type strains of Mycoplasma canis, M. bovirhinis and M. dispar and ten recent M. canis isolates from cattle were determined. Metabolism of a range of sugars and organic acids by M. dispar was detectable by measurement of oxygen uptake. Organic acids were not utilized by M. bovirhinis or M. canis, and there was no oxygen uptake during metabolism of glucose or other sugars, as monitored by a pH-change method. The M. canis strains varied in their ability to metabolize sugars; seven of the isolates from cattle had the distinctive ability to metabolize sucrose, and one isolate, plus the type strain (from a dog), metabolized N-acetylglucosamine. The M. bovirhinis strain metabolized maltose. However, all the test strains oxidized glycerol at high rates and with a high affinity. Oxidation of glycerol has been reported for other mycoplasmas from the bovine respiratory tract and leads to the production of hydrogen peroxide, a potential virulence factor.     

牦牛肺炎病原多杀性巴氏杆菌和殊异支原体的分子鉴定与分析

青海省刚察县某牦牛产业基地牦牛发生咳嗽,鼻孔有分泌物流出,呼吸困难等症状的疾病。为快速确诊发病牛的致病原因,及时防控和治疗,从发病和病死的牦牛中,采集鼻腔拭子和肺脏、胸腔积液,利用PCR方法进行分子鉴定与分析。结果显示:此牛场中引发肺炎的病原是A型多杀性巴氏杆菌和殊异支原体两种病原体混合感染,测序结果显示5个有效样品中鉴定的为完全相同的病原,P.multocida-GC1的Kmt基因与参考序列中国株CP031554/3/2/1同源性在99%以上,M.dispar-GC1与参考序列的16S rRNA基因同源性在97%以上。同时在遗传进化角度再次分析确认了鉴定的P.multocida与中国和俄罗斯的A型多杀性巴氏杆菌聚成一大支;鉴定的M.dispar与殊异支原体聚为一支。结果表明,青海刚察地区牦牛存在感染多杀性巴氏杆菌和殊异支原体的潜在风险,该研究为牦牛呼吸道疾病与肺炎的病原学调查和病原的流行病学调查提供了参考数据。     

羊附红细胞体体外培养最佳条件的探索

近几年,内蒙古自治区锡林郭勒盟多地出现了一种羊病,发病率和死亡率都较高,最终诊断为羊附红细胞体病。为给羊附红细胞体（Mycoplasma ovis,M. ovis）深入研究奠定基础,参考支原体培养基,进行配方改良,对M. ovis的最佳培养条件进行了初步研究。结果表明：应用实验室自制ZP-S-BS培养基,48 h更换半量培养基,37 ℃培养效果最佳。感染M. ovis红细胞用ZP-S-BS培养能维持长时间存活状态,洗脱的M. ovis用ZP-S-BS培养仍可连续培养260 d,且能缓慢增殖。     

Mycoplasma Dispar As a Causative Agent in Pneumonia of Calves

Three field strains of Mycoplasma dispar were inoculated, by aerosol inhalation, into a total of eight naturally-born, colostrum-deprived calves. All three strains produced macroscopic pneumonia, each in one calf. Histopaithologically an exudative bronchitis accompanied by moderate interstitial cell proliferations was found.Reisolation studies indicated that the entire respiratory tract is the natural habitat of Mycoplasma dispar, which apparently does not spread via the blood stream.     

Transmission of Mycoplasma dispar among calves collected and reared for beef production

Fortytwo calves, 28 to 117 days old, were collected from 23 dairy farms and transported in a lorry, allowing direct contact between the calves, to 8 calf rearing farms. The average transport time per calf was 4.5 h, ranging from 0.3 to 12.8 h. The calves were sampled by nasal swabbing for mycoplasmas first before loading and then immediately after transport. Thirteen of the calves were transferred to farm I. They were placed in individual pens in a separate room to themselves, and were sampled at intervals for a period of 4 weeks.Ten of the 42 calves (23.8 %) originating from 5 of the source farms were found initially positive for M. dispar with titers > 4 log₁₀ ecu; 3 of these 10 calves were delivered to farm I and 7 calves to 6 others of the 8 receiving farms. Three initially infected calved delivered to farms I continued to be positive throughout the follow-up period; among the 10 initially negative calves the frequency of detected infection, and the geometric mean titer (within parenthesis), developed so that on days 1, 7, 14, and 28 the figures were: 2 (2.5), 8 (4.3), 9 (4.7), and 10 (5.5), respectively.After transport 3 initially negative calves were found positive with low titers. Two of them were placed on farm I. In one of them positivity proved to be only transient; the case seems to represent a phenomenon of transfer of mycoplasmas without establishment of infection. In contrast, at least 4 (possibly 7) calves, negative both before and after transport–ascribed above to the group of 10 initially negative calves arriving on farm I–had in all likelihood caught the infection during the transport. Two of the 10 calves most likely caught the infection on the farm; for 3 calves the evidence was equivocal as to the 2 alternatives.Seven of the 42 calves (16.7 %) were found to be initially infected with M. bovir-hinis, 2 of the 42 with Acholeplasma laidlawii. Among the 13 calves transported to an reared on farm I, 8 were found to be positive at least once for M. bovirhinis during the study. Colonisation by this mycoplasma was partly detected only intermittently and the detectable prevalence among the 13 calves at its highest was only 38.5 %.     

Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar

Inoculation of tracheal organ cultures from bovine foetuses with Mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20-22 days later by lifting and detachment of overlying epithelium. The effect was associated with large numbers of M. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. Ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. In contrast, M. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. The cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma.     

牛支原体的分离鉴定及其对体外药物的敏感性分析

为探讨兽医临床治疗牛支原体肺炎常用药物的有效性,采用形态观察、分子生物学等方法对病原菌进行鉴定,共获得4株牛支原体,继而对分离菌株进行耐药性检测。结果表明,4株牛支原体均对大环内酯类抗生素耐药,对氟喹诺酮类及四环素类抗生素相对敏感。实验结果为兽医临床对牛支原体肺炎的治疗具有一定的指导意义。     

The prevalence and level of colonisation by Mycoplasma dispar and other mycoplasmas on calf rearing farms

The prevalence and level of colonisation by respiratory mycoplasmas, especially by Mycoplasma dispar (M. dispar), in calves on 24 Finnish calf rearing farms were studied by using nasal swabs. A minimum of 5 calves from each farm was examined and 91.3 % of the 206 calves examined were 1 to 5 months old. Nine herds were selected on account a diagnosed respiratory disease problem, the others without anamnestic knowledge of their health condition.All 24 farms and 96.1 % of the calves examined were found to harbour one or more species of mycoplasma. M. dispar, M. bovirhinis and Acholeplasma laidlawii were isolated from 23, 19 and 3 farms or from 91.7, 51.9 and 3.4 % of the calves investigated, respectively.The prevalence for M. dispar was 97.8 % and the geometric mean titer 4.9 log₁₀ color changing units (ccu) among 1- to 5-month old calves belonging to the infected farms. The prevalence in the age-group of 6- to 12-month old calves was significantly lower (40 %). No significant difference was found in the level of colonisation by M. dispar among the one-month age-groups of the 1- to 5-month old calves. A high degree of colonisation among 1- to 2-month old calves was indicative of the high ability of M. dispar to spread and colonise the respiratory pathway of young calves in the conditions described. The same state still prevailing in calves aged 4 to 5 months supported the concept of a relatively long duration of a heavy colonisation by this mycoplasma. The prevalence of M. bovirhinis among 1-to 5-month old calves was lower than that of M. dispar. Any decrease in the level of colonisation of M. bovirhinis could not be demonstrated in older calves. The titer levels of both M. dispar and M. bovirhinis as well as the prevalence of M. bovirhinis were significantly higher in herds having a current respiratory disease problem than in healthy or mildly affected herds. The same relationship applied to the husbandry method of common pens as compared with individual pens. A causal relationship of increased colonisation level, either as cause or effect, to the respiratory disease is suggested, as well as a probably more indirect one to the common pen husbandry type.     

甘肃地区绵羊支原体病原菌的分离培养

在甘肃省5个地区采集了100份绵羊血清样本,并对100份样品进行了血清学及病原菌分离培养试验。血清学检测初步鉴定为14份阳性,其中11份来自于同一地区。然后对两例疑似患病羊做病原分离鉴定以及微生物学诊断和PCR鉴定,并对病原菌进行革兰氏染色。本研究成功地从病羊身体中分离出细小、纤细的呈球形、双球形、线状、螺旋状、半月状和短杆状等形态的绵羊肺炎支原体。     

支原体PCR检测方法的建立和应用

依据GenBank中登录的常见细胞污染支原体16S rRNA基因序列,选择高度保守的区域设计引物,建立了一种快速灵敏的支原体PCR检测方法.该方法可以特异性检测出6种支原体,并能敏感的检测到10个拷贝数的目的基因.采用建立的PCR方法和传统的培养法对23个不同样品(细胞、血清、疫苗成品、半成品)进行检测,符合度100％.该方法的建立可大大缩短疫苗生产过程中支原体检验所需时间.     

鹅败血支原体及滑液囊支原体感染的血清学调查

本研究对珠江三角洲部分地区鹅群的185例血清样品，进行败血支原体、滑液囊支原体感染的平板凝集试验，检测结果表明：鹅败血支原体的感染率为29.7%，滑液囊支原体的感染率为19.5%，两者的混合感染率为14.6%。     

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。     

D型多杀性巴氏杆菌和绵羊肺炎支原体引起山羊的混合感染

2018年9月广西某羊场部分山羊发生流涕、咳嗽、呼吸困难和体温升高等临床症状的疾病,为确诊发病原因并提供治疗方案,采用病原分离培养、PCR扩增鉴定的方法进行诊断,并对分离菌进行生化鉴定、致病性试验和药敏试验。结果显示,病料在血平板有圆形的小菌落生长,革兰阴性小球短杆菌,而在PPLO培养基上不生长;病料的PCR扩增结果显示绵羊肺炎支原体和多杀性巴氏杆菌均为阳性;所分离到的病原菌经生化鉴定,该菌符合多杀性巴氏杆菌的特性;用多杀性巴氏杆菌种属和D型多杀性巴氏杆菌特异性引物扩增为阳性;致病性试验显示该菌对小鼠有很强的致病性;药敏试验显示该菌对头孢他啶、头孢噻肟、氧氟沙星高度敏感,对复方新诺明、强力霉素、红霉素、青霉素为耐药。结果表明该病是由绵羊肺炎支原体和D型多杀性巴氏杆菌混合感染引起。     

氟喹诺酮类药物压力下鸡毒支原体gyrA基因突变特征分析

按常规方法提取鸡毒支原体参考株(S6-10)、疫苗株(F14-6)及在氟喹诺酮类药物压力下体外筛选出来的耐药株的基因组DNA，扩增各菌株DNA旋转酶gyrA基因片段，并克隆、测序，利用DNASIS分析软件对测序结果进行分析。结果表明，S6-10和F14-6在恩诺沙星或环丙沙星压力下均筛选出不同程度的耐药菌，而且恩诺沙星致鸡毒支原体发生耐药性突变的机率高于环丙沙星，S6-10株耐药突变频率高于F14-6。S6-10筛选出来的耐药菌株发生突变的位置在DNA旋转酶gyrA亚基的第83位(相对于大肠杆菌gyrA亚基中的位置，以下同)和87位上，取代模式为Ser83→Ile、Glu87→Gln，高水平耐药株(Se32M，MIC≥128)除了在第83位发生突变外，在第82位还增加一突变位点(Asp→Gly)；F14-6筛选出来的高水平耐药菌株(Fe32M)在DNA旋转酶gyrA亚基的第83、87位发生了双位点突变(Ser83→Ile，Glu87→Gly)，而低水平耐药株(Fe8M、Fc8M)在gyrA亚基未发生任何位点突变。     

恩诺沙星与其他抗菌药对鸡毒支原体的体外联合抑菌试验

采用2倍稀释法测定了恩诺沙星及其他8种抗菌药对鸡毒支原体BG44T株的最小抑菌浓度（MIC）,再以棋盘法测定恩诺沙星分别与其他8种抗菌药不同联合对鸡毒支原体BG44T株的敏感性。结果显示：恩诺沙星、替米考星、泰乐菌素、吉他霉素、林可霉素、沃尼妙林、泰妙菌素、氯霉素及氟苯尼考对鸡毒支原体BG44T株的MIC分别为0．063、0．004、0．016、0．063、16、〈0．004、0．008、8、8μg／mL。在8种不同联合用药对鸡毒支原体BG44T株的药敏试验中,恩诺沙星＋替米考星、恩诺沙星＋泰乐菌素、恩诺沙星＋吉他霉素、恩诺沙星＋林可霉素、恩诺沙星＋沃尼妙林、恩诺沙星＋泰妙菌素联合表现出相加作用,恩诺沙星＋氯霉素、恩诺沙星＋氟苯尼考表现出拮抗作用。     

山羊传染性胸膜肺炎支原体和绵羊肺炎支原体对抗菌药物敏感性的研究

测定了环丙沙星、氧氟沙星、单诺沙星、红霉素、罗红霉素、泰乐菌素、泰妙菌素、四环素等8种药物对羊肺炎支原体两个标准株Y-98和Y-goat的体外抑菌浓度以及红霉素与氧氟沙星、泰乐菌素对Y-goat和四环素与氧氟沙星、泰乐菌素对Y-98的联合药敏作用.结果表明,这8种抗菌药物对Y-goat和Y-98的MIC(μg/mL)分别为:环丙沙星0.223、0.002 23,氧氟沙星0.281、0.014 0,单诺沙星0.136、0.014 0,红霉素0.021 8、无效,罗红霉素0.032 7、无效,泰乐菌素0.042 2、0.039 0,泰妙菌素0.021 7、0.052 0,四环素0.195、0.052 0.红霉素与氧氟沙星的联合药敏指数为1,是相加作用;红霉素与泰乐菌素对Y-goat的联合药敏指数为1.5,是无关作用;四环素与氧氟沙星、泰乐菌素对Y-98的联合药敏试验指数均为0.375,是协同作用.     

1株鸡毒支原体的分离鉴定及体外敏感中药的筛选

本研究旨在对山东泰安地区鸡毒支原体的流行菌株进行分离培养及鉴定和纯化,并针对该分离株筛选体外敏感的中药,为后续进一步研究提供材料。采用液体培养法与固体培养法从病鸡组织样品中分离、培养和纯化鸡毒支原体,并通过瑞士染色和姬姆萨染色、血清学方法及分子生物学方法对分离株进行初步鉴定,在此基础上,采用微量稀释法测定8种中药对分离菌株的最小抑菌浓度（MIC）,从而筛选到对分离菌株敏感的中药。结果显示,分离菌株在鸡毒支原体液体培养基中增殖后,培养基颜色由红变黄且呈半透明状态,在固体培养基中培养后呈典型的"煎蛋"样儿菌落,均符合鸡毒支原体培养特性;瑞士染色和姬姆萨染色结果显示,菌体形态符合鸡毒支原体的特征;血清学鉴定结果显示,分离株与鸡毒支原体阳性血清发生凝集;分子生物学鉴定结果显示,所扩增的核酸序列与鸡毒支原体匹配度高达99%;MIC测定结果显示,中药黄连和黄柏对分离的鸡毒支原体的抑菌活性较强,属敏感范畴,其MIC分别为≤0.98和0.39 mg/mL,而中药金荞麦和鱼腥草对鸡毒支原体中度敏感,MIC均≥125 mg/mL,板蓝根、白鲜皮、当归、艾叶对鸡毒支原体均不敏感。综上所述,本研究成功分离到1株鸡毒支原体,并且筛选出4种对该菌株敏感的中药,分别为黄连、黄柏、金荞麦和鱼腥草,为后期进一步研究防治鸡毒支原体病的中药组方时提供参考依据。     

板黄口服液对牛支原体的敏感性试验

为了研究复方板黄口服液对牛支原体的体外抑制作用,以指导临床合理用药,试验采用试管二倍稀释法测定最小抑菌浓度。结果表明：该制剂对牛支原体有抑制作用,其最小抑菌浓度为500 mg/mL,说明纯中药制剂板黄口服液对牛支原体有较好的抑制作用。     

唾液乳杆菌对鸡毒支原体感染肉鸡生长性能及肺损伤的影响

本研究旨在探究唾液乳杆菌（Lactobacillus salivarius）对鸡毒支原体（Mycoplasma gallisepticum,MG）感染肉鸡生长性能及肺损伤的影响。选用1日龄AA肉鸡240只,按试验要求随机分为6组,包括对照组（Con）、低剂量唾液乳杆菌组（L）、高剂量唾液乳杆菌组（H）、MG感染组（MG）、MG感染+低剂量唾液乳杆菌组（MG+L）和MG感染+高剂量唾液乳杆菌组（MG+H）。Con组、MG组饲喂基础饲料,L组、MG+L组饲喂添加10⁸ CFU·kg^-1唾液乳杆菌的基础饲料,H组、MG+H组饲喂添加10⁹ CFU·kg^-1唾液乳杆菌的基础饲料,7日龄时给与MG组、MG+L组和MG+H组进行MG感染攻毒,饲养42 d后,通过分析各组鸡的体重、饲料转化率、肺部MG定植量、肺部病理损伤、肺部炎性损伤相关蛋白表达量及促炎性细胞因子含量来评价唾液乳杆菌缓解MG感染所致肉鸡生长性能下降及肺损伤效果。结果表明,MG感染极显著降低肉鸡体重（P<0.01）,并极显著提高饲料转化率（P<0.01）,而饲料中添加低剂量唾液乳杆菌和高剂量唾液乳杆菌均能极显著缓解MG感染所致肉鸡体重降低（P<0.01）,并极显著改善饲料转化率（P<0.01）。饲料中添加低剂量唾液乳杆菌和高剂量唾液乳杆菌均能极显著降低MG肺部定植量（P<0.01）;明显改善MG感染所致肺部病理损伤;极显著降低MG感染所致炎性损伤相关蛋白（TLR2、HMGB1、p-p65/p65、NLRP3、Pro-Caspase-1/Caspase-1）表达（P<0.01）;极显著降低MG感染所致促炎性细胞因子（TNF-α、IL-1β、IL-6、IL-8）含量（P<0.01）。综上所述,饲料中添加唾液乳杆菌可以显著缓解MG感染所致肉鸡生长性能下降及肺损伤,该株唾液乳杆菌具有较大的应用潜力预防肉鸡感染MG。     

Detection of Mycoplasma pulmonis in laboratory rats and technicians

Five species of mycoplasma are associated with several rat diseases. Mycoplasma pulmonis is the most important and most studied, possibly causing disease in rats and undermining the validity of laboratory experiments. M. pulmonis was isolated in 144/240 laboratory rats and identified by PCR in 155/240. This species was also detected in 12 human individuals (technicians of a laboratory animal house hold) in contact with these rats. The results were confirmed by sequencing of DNA products. Mycoplasma species are host specific; however, M. pulmonis was identified in humans, suggesting a case of unspecific colonization. Statistical analysis shows a greater risk for M. pulmonis colonizing individuals who are exposed to infected rats in animal facilities than individuals who do not. The detection of M. pulmonis in humans indicates a new status for this mollicute mycoplasmas in animal-holding facilities.