致犊牛肺炎牛支原体新疆北屯株的分离鉴定及oppF基因序列分析

为确诊新疆北屯市某牛场致犊牛肺炎死亡的病原,试验无菌采集2头病死牛肺脏组织分离培养牛支原体,并进行特异性PCR鉴定及oppF基因的序列比对。结果表明:分离获得2株牛支原体,分别命名为M.bovis BT-1和M.bovis BT-2;2株分离株的菌落形态呈典型的"煎蛋样";PCR扩增出牛支原体oppF基因片段,与预期大小一致;2株分离株的oppF基因与国际牛支原体标准株PG45的同源性分别为97.3%和97.8%;与HB0801、CQ-70W在同一进化分支上,亲缘关系较近。     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

牛支原体贵州株表面膜蛋白p81基因生物信息学分析

为了解牛支原体(Mycoplasma bovis)贵州株表面膜蛋白p81基因的生物信息学特征,对2株贵州分离株p81基因进行克隆测序,应用DNAStar 7.1、Mega 5.0、Protparam、Protscale、IEBD等软件进行序列分析。结果显示,2株M.bovis贵州分离株培养物DNA样本均能PCR扩增出2 187 bp的特异性条带,序列编码728个氨基酸,该分离株与标准株PG45进化关系最为接近,说明牛支原体p81基因具有良好的保守性。p81蛋白共存在5个N糖基化位点,59个丝氨酸、29个苏氨酸及11个酪氨酸可能被磷酸化。p81蛋白B细胞抗原表位分析显示该蛋白具有良好的抗原性。研究结果表明,p81蛋白具有作为亚单位疫苗和诊断试剂靶标蛋白的优势。     

牛支原体贵州株TU基因的克隆及序列分析

为了解牛支原体(M.bovis)贵州株GZ1、GZ2 TU基因的生物信息学特征,试验对牛支原体贵州株TU基因进行克隆测序和序列分析,应用DNAStar软件对TU基因推导氨基酸序列蛋白亲水性、表面可及性、骨架柔韧性及抗原指数进行预测和分析。结果表明:TU基因克隆测序基因片段长度为1 009 bp,与预期相符。TU基因序列分析显示,牛支原体GZ1株与PG45标准株同源性为99.1%,与GZ2株同源性为99.0%。基因变异性显示,参考牛支原体标准株PG45,GZ1株在第55,134,191,257,389,512,674位发生了突变,在第178,951,962位发生了缺失,GZ2株仅在273位发生了突变。进化树分析显示,GZ1株与国内分离株属同一分支,进化关系近。GZ1株与GZ2、PG45株属不同分支,并且与国外的分离株进化关系较远。TU基因B细胞抗原表位预测结果显示,该蛋白亲水性、表面可及性、骨架区柔韧性都较好,抗原指数高,且分布相对均匀。该蛋白整体区域抗原优势明显,具有良好的抗原性。说明牛支原体TU基因相对保守,各地区分离株存在一定的进化关系。     

牛支原体pdhc基因的克隆、表达及其亚细胞定位

试验旨在研究牛支原体(Mycoplasma bovis,M.bovis)武威株二氢硫辛酰胺转乙酰酶(PDHc-E2)基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因(登录号:CP002058.1)设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a(+)中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta(DE3)感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体(M.agalactiae)同源性为90.9%~91.2%,与加利福尼亚支原体(M.californicum)ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1∶100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。     

牛支原体湖北株的分离鉴定及其oppF基因的进化分析

牛支原体(Mycoplasma bovis)是引起犊牛肺炎、关节炎和成牛乳房炎的主要病原之一。从湖北恩施暴发牛支原体的肉牛场(70头引种肉牛出现20%的死亡)分离得到一株牛支原体,命名为HB2015,并对其进行了保藏(保藏号:CCTCC M 2016559),进化分析结果显示该菌株与M.bovis NM2012内蒙株亲缘关系较近,为牛支原体的防控提供了理论依据。     

牛支原体能量耦合因子转运蛋白S组件MbBioY摄取外源生物素的功能分析

牛支原体(M.bovis)感染与生存依赖于外界微环境提供的各类营养代谢因子。开展M.bovis外源生物素摄取的机制研究,将对M.bovis防控具有极其重要的意义。通过对M.bovis PG45 MbBioY(MBOVPG45＿0349)基因进行系统发育分析、分子对接分析,筛选M.bovis PG45 MbBioY突变株,比较PG45和PG45ΔMbBioY的生物素敏感性,异源构建MbBioY功能性克隆验证其功能。结果表明,PG45染色体上存在一种崭新的生物素膜转运蛋白,全长996 bp, GC含量30.95%,蛋白长度为332个氨基酸,是由7个跨膜结构域组成的能量偶合因子(ECF)转运蛋白S成分MbBioY。进一步验证后发现,当M.bovis缺失MbBioY会影响其生长,表现出生物素代谢营养缺陷的表型。通过异源敲入生物素代谢营养缺陷大肠杆菌MG1655,表明MbBioY单独存在时,就能够恢复生物素代谢营养缺陷大肠杆菌缺失株的生理功能。初步证明,MbBioY编码的是能量偶合因子(ECF)转运蛋白的S成分具有摄取外源生物素的能力,该研究结果将补充细菌生物素代谢调控的多样性和复杂性,为M.b...     

牛支原体pdhc基因的克隆、表达及其亚细胞定位

试验旨在研究牛支原体（Mycoplasma bovis,M.bovis）武威株二氢硫辛酰胺转乙酰酶（PDHc-E2）基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因（登录号：CP002058.1）设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a（+）中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta（DE3）感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735 bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体（M.agalactiae）同源性为90.9%~91.2%,与加利福尼亚支原体（M.californicum）ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29 ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1：100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。     

牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

试验旨在确定牛支原体P48基因的免疫原性，为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象，运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因，构建原核表达载体pET-32a （+）-P48，转化大肠杆菌BL21（DE3）感受态细胞，在诱导剂ITPG的诱导下获得重组蛋白P48，纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体，运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明，试验成功构建原核表达载体pET-32a （+）-P48，重组蛋白P48大小约为66 ku，纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应，血清抗体滴度达到较高水平（D_{450 nm}值为1.126）。Western blotting结果显示，抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应，表明P48重组蛋白具有良好的免疫原性与反应原性，可作为牛支原体新型疫苗的候选基因，且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高，亲缘关系较近。     

Molecular and antigenic characterization of a Mycoplasma bovis strain causing an outbreak of infectious keratoconjunctivitis.

An unusually high incidence of infectious keratoconjunctivitis followed by pneumonia and arthritis was observed in beef calves of a managed herd. No Moraxella spp. or bacteria other than Mycoplasma spp. were obtained from conjunctival and nasal swabs. A strategy was designed for characterization of bovine mycoplasmas at species and strain level on the basis of a combination of molecular tools and the immunoblotting method. The strategy made it possible to rapidly assign the bacterium responsible for this outbreak to one of the phylogenetic clusters of bovine mycoplasmas delineated in this study and then to identify it as Mycoplasma bovis. The strain, designated Sar 1, showed a 100% 16S rDNA sequence identity with two European strains (120/81 and MC3386) isolated in Germany and Ireland, respectively, and hosts a vsp gene analog to the vspA, vsp422-4, and vsp422-8 genes of the M. bovis reference strain PG45T and of the field strain 422. The use of a cross-reactive rabbit serum developed against the Mycoplasma agalactiae immunodominant antigen P48 confirmed the molecular findings. The immunological response of calves against M. bovis was also investigated. This is the first report on the occurrence of M. bovis on the Island of Sardinia (Italy).     

Genetic and antigenic characterization of the surface lipoprotein P48 of Mycoplasma bovis

The presence of a membrane lipoprotein homologous to the P48 of Mycoplasma agalactiae was investigated in different Mycoplasma bovis isolates selected by geographical locations and biological properties. Its potential as a diagnostic tool was also discussed. The presence of a specific signal observed in all M. bovis field isolates probed with a rabbit antiserum raised against the M. agalactiae recombinant P48 demonstrated that this protein is structurally and antigenically conserved within the M. bovis cluster. No signal was detected when testing six different mycoplasma species found in cattle. The p48 gene was identified by PCR approach and partially sequenced. Full length gene sequence was obtained by direct bacterial chromosome sequencing. Five UGAs were selectively mutated into UGG and the full length mutated gene, lacking the signal peptide, was cloned and expressed in Escherichia coli. The purified recombinant antigen (r-P48) was evaluated as a potential marker of infection using a panel of 86 well-characterized sera from experimentally and naturally infected cattle. Specific IgM antibodies were detected within 6-9 days after experimental infection followed by an IgG response lasting from the third/fourth week after contact. Although antibody titers were well below those observed in sheep or goats infected with M. agalactiae, results suggest that M. bovis r-P48 can be used as a specific marker of infection.     

牛支原体新基因(P18)的克隆表达及活性鉴定

牛支原体(M.bovis)可以引起犊牛肺炎、关节炎、乳腺炎、角膜结膜炎等疾病,是一种牛的重要呼吸道病原体,目前M.bovis粘附宿主细胞的机制还不明确。研究表明,M.bovis表面存在的多种蛋白与致病菌在宿主体内的侵袭力与传播能力有关。我们通过分离表达多个M.bovis基因,最终获得了一个表达纤溶酶原结合蛋白的新基因,并命名为P18。该基因表达大小约为67ku的重组蛋白。通过western blot鉴定,重组表达的P18蛋白可以被M.bovis抗体识别。进一步的试验表明,P18蛋白还具有纤溶酶原结合活性,从而推测该基因表达的蛋白可能是M.bovis的一个粘附相关因子。     

牛支原体新疆分离株P48基因克隆及分子特征分析

本研究旨在分析牛支原体P48基因的序列特性、蛋白的结构和功能。根据GenBank中PG45菌株P48基因序列（GenBank登录号：CP002188.1）设计特异性引物,运用Overlap-PCR扩增获得P48基因全长,其目的片段连接到pMD19-T载体上并进行测序,利用生物信息学方法分析和预测其核苷酸序列及其编码蛋白的理化性质、结构功能（信号肽、跨膜结构、磷酸化位点、糖基化位点、二级结构和三级结构）。结果显示,分离株P48基因序列为1 407 bp,与Mb PG45国际标准株的同源性为100%,聚类于一支;P48基因编码467个氨基酸残基,组成一个无跨膜、稳定的亲水性蛋白;P48蛋白存在信号肽,有44个潜在的磷酸化位点和1个糖基化位点,其二级结构是混合型,其中α-螺旋所占比例最高（41.76%）,无规则卷曲次之（37.69%）。功能预测显示,在信号转导、胁迫应答、受体等方面该蛋白存在较高概率。本研究成功克隆了分离株P48基因片段,其编码的蛋白是一个稳定可溶、亲水性蛋白,为分析分离株P48基因的特性和功能提供了一定的理论基础和科学依据。     

牛支原体、巴氏杆菌A型和化脓隐秘杆菌多重PCR快速检测方法的建立

本试验旨在建立一种可同时鉴别牛支原体、巴氏杆菌A型和化脓隐秘杆菌的多重PCR方法。分别针对多杀性巴氏杆菌A型特异的hyac-hvaD基因区段、化脓隐秘杆菌的16SrRNA基因上保守区段和牛支原体的UvrC基因设计特异性引物,多重PCR的最佳扩增条件确定为：95℃ 10min预变性;95℃ 1min,56℃ 50s,72℃ 1min,循环30次;72℃ 210min延伸。结果表明,该多重PCR方法可同时扩增出以上三种致病菌的特异性片段,不能扩增出其他病原菌的相关片段;对多杀性巴氏杆菌A型、化脓隐秘杆菌和牛支原体的最低检测浓度分别为8×10^5CFU／mL、8×10^5CFU／mL和4×10^6CFU／mL。同时用该方法检测了牛支原体肺炎患牛的鼻拭子与肺组织,发现12h预增菌后,肺组织检测与牛支原体培养的阳性符合率为92％。对临床样本进行牛支原体分离培养需要3-4d时间,而采用多重PCR方法检测12h预增菌则能在24h内出结果。该多重PCR方法显著加快了临床诊断速度,具有推广应用价值。     

Cloning and characterization of a Moraxella bovis cytotoxin gene

OBJECTIVE: To identify the Moraxella bovis cytotoxin gene. PROCEDURE: Hemolytic and nonhemolytic strains of M. bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M. bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. RESULTS: Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M. bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M. bovis cytotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M. bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis.     

一株牛支原体的分离鉴定及致病性初步研究

对采集到的疑似牛支原体肺炎肺组织病料进行病原的分离,并对分离株进行形态学、生化和分子生物学鉴定,结果显示成功分离获得1株牛支原体,命名为NM001。该分离株的菌落形态呈典型的“荷包蛋状”,不能发酵葡萄糖,不能水解精氨酸,不分解尿素。PCR能够扩增出牛支原体特异的P48基因条带,16S rRNA基因序列与Ningxia-1序列同源性为99.03%。将该分离株接种2头6月龄犊牛均出现明显的临床症状,剖检后胸腔中少量淡黄色渗出液,肺脏出现肉样实变。试验结果表明,分离到的牛支原体NM001株对牛具有较强的致病性,为牛支原体攻毒模型的建立奠定了基础。     

In vitro and in vivo evaluation of lysozyme extracts of virulent Mycobacterium bovis in guinea pigs and calves

Extracts of Mycobacterium bovis ATCC 19210 were prepared from cells following treatment with acetone 3 times, ethyl alcohol-ether 3 times (1:1, v/v), and chloroform 3 times. Cells were dried and suspended in 0.05M Tris-HCl (pH 7.5) containing lysozyme (1 mg/50 mg of dried cells). One aliquot of the lysozyme extract was filter-sterilized and 1 aliquot of the lysozyme extract was autoclaved. Delayed-type hypersensitivity responses elicited in sensitized guinea pigs, using the filter-sterilized lysozyme extract, were significantly greater than responses elicited using the autoclaved lysozyme extract (P less than 0.01). The filter-sterilized lysozyme extract and a purified protein derivative (PPD) of M bovis, at equal protein concentrations, elicited comparable delayed-type hypersensitivity responses in sensitized guinea pigs. Significant differences were not detected between the mean enzyme-linked immunosorbent assay (ELISA) values on sera collected from calves before exposure to M bovis, using each of the lysozyme extracts or the M bovis PPD (P greater than 0.05). Significant differences were detected when ELISA values obtained using each of the antigens on post-exposure serum were compared with ELISA values on serum collected from calves before exposure to M bovis (P less than 0.01). Differences were not detected in mean ELISA values on sera from cattle collected 12 months after exposure to M bovis, using each of the lysozyme extracts or M bovis PPD (P greater than 0.05); however, 8 of 8 calves were identified as positive on ELISA, using the filter-sterilized lysozyme extract, 7 of 8 calves were positive, using M bovis PPD, and 7 of 8 calves were positive, using the autoclaved lysozyme extract.     

On the aetiopathogenesis of Mycoplasma pneumonia in calf

A pure culture of Mycoplasma (M.) bovis was isolated from calves with respiratory disease, exhibiting the picture of lymphohistiocytic proliferative pneumonia with presence of eosinophil plasmatic cells. A mixed infection of M. bovis and Pasteurella (P.) multocida was demonstrated in calves with exudative pneumonia. Both M. bovis and Haemophilus (H.) somnus were recovered from calves with necrotic pneumonia. All 3 organisms--M. bovis, P. multocida, and H. somnus--were present in cases of exudative-necrotic pneumonia. It was also shown that M. bovis played a primary role in the aetiopathogenesis of respiratory diseases caused by mixed infections.