Establishment of a Collection and Detection Technology of Mycoplasma hyopneumoniae in Aerosol

ObjectiveThis study was to establish a simple method for collecting and detecting Mycoplasma hyopneumoniae （Mhp） in aerosol. MethodBased on the mechanisms of liquid impinger and filtration sampler, a double concentration aerosol sampler was designed for collecting Mhp aerosol. Firstly, the collection was performed in a closed environment full of artificial aerosol of Mhp. Secondly, collection efficiency was detected by real-time PCR. Thereafter, the clinical feasibility of the designed equipment was tested by collecting aerosol samples in different pig herds. In one assay, the samples were collected at different times from one pig house challenged with Mhp. In another assay, the samples was collected from the delivery room, nursery and fattenning house of a MPS outbreak farm as well as a Mhp infection positive pig farm without obvious clinical symptoms. All the aerosol samples were then detected by real-time PCR or nested PCR. ResultThe collection efficiency of the designed bioaerosol sampler was （37.04±6.43） %, Mhp could be detected 7 d after intratracheal challenge with pneumonic lung homogenate suspension. Aerosol samples of 11 pig houses from the two Mhp positive pig farms with or without clinical symptoms all showed a positive result of PCR, the positivity rate was 100%. ConclusionA high sensitive collecting and detecting technology of aerosol was successfully established, which can be applied to clinical detection of Mhp in aerosol.

Effect of Osmotic Pressure on Mycoplasma hyopneumoniae Strain 168

[Objective] This study aimed to investigate the effect of hypertonic solution on the morphology,size,incubation time and cell viability of Mycoplasma hyopneumoniae（Mhp）.[Method] Mhp was stimulated by using NaCl hypertonic solution with a final concentration of 1.8% for 1,2,4,6 and 8 min,respectively.After staining and microscopic examination,the particle size and CCU（color change unit） were determined,and PCR identification was conducted.[Result] After treated with hypertonic solution for different time,Mhp was shrunken and turned round,the particle size was reduced,and the cell number rapidly decreased or even disappeared after 2 min.CCU was reduced by a gradient,and the observation time was extended for 1 d.The target band of Mhp could be amplified from samples after treated with hypertonic solution for different time.After treated with 1.8% NaCl solution for 1-6 min,Mhp changed in the morphology and size but still had viability.[Conclusion] This study provided reference data for exploring the entrance of Mhp into blood circulation after intramuscular injection and a new immunization route of swine Mycoplasma pneumonia vaccine.

Advances in the Detection of Mycoplasma hyopneumoniae by Polymerase Chain Reaction (PCR) Technology

Mycoplasma hyopneumoniae is an important pathogen causing Mycoplasmal pneumonia of swine, which generally causes secondary infections and mixed infections, thus seriously threats the development of swine industry and resulting in huge economic losses. Using PCR technology has very important significance to the correct diagnosis of Mycoplasmal pneumonia at the early stage. In this paper, specific target genes of Mycoplasma hyopneumoniae, methods for clinical sample collection, key technical factors of DNA sample processing method, and the research progress, main advantages and disadvantages, and application of general PCR technology, multiple PCR technology, nested-PCR technology, real-time fluorescence quantitative PCR technology, gene chip detection technology and loop-mediated isothermal amplification in detection of Mycoplasma hyopneumoniae were summarized, which provided convenience for the effective diagnosis and prevention of Mycoplasmal pneumonia of swine.

Development and Application of a Quantitative Competitive PCR Assay for Detecting Mycoplasma hyopneumoniae

[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The competitive template, which carried the same primer binding site with the target fragment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa（X-axis）, and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate（Y-axis）. Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit（CCU） were treated as the abscissa（X-axis）, and the copy numbers of Mhp were treated as the ordinate（Y-axis）, so the standard curve was generated. It was found that the copy number of Mhp was highly correlated to CCU. [Conclusion] A quantitative competitive PCR assay was successfully established for the rapid detection of Mhp in culture.

Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Comparative Analysis for the Detection and Monitoring of Mycoplasma hyopneumoniae Infection by Nested PCR(n-PCR) and Real time PCR(q-PCR) from Field Swine Herds

[Objective] 303 nasal swabs samples were collected from pigs in farms located in Taizhou city, Jiangsu Province, China from March to December 2012 for the purpose of detecting the presence of Mycoplasma hyopneumoniae, the primary agent of Enzootic porcine pneumonia （EPP） in pig herds using the nested PCR and Real time PCR techniques. [Method] Nasal swabs were collected from pigs of different ages＇ i.e. 7, 14, 21, 28, 30 and 35 days old, soaked in sterile 1 xPBS overnight at 4 ℃ and DNA extracted using the TIANamp（R） bacterial DNA kit. The DNA samples underwent amplification under the Mhyo 183 q-PCR and P36 primer Nested PCR systems. [Result] With the Nested PCR assay, 38 （12.5%） out of 303 samples tested positive for the presence of M. hyopneumoniae; with the real time PCR assay 152 （50.2%） tested positive for M. hyopneumoniae. The two assays matched to positively detect Mhyo in 22 （7.3%） samples and again matched in 127 （41.9%） samples negative for Mhyo infection. The pattern of infection in both assays was similar where 7- and 35-day-old piglets in both assays had the highest rates of infection i.e. 15.6% and 18.4% for n-PCR and 53.1% and 56.6% for q-PCR for 7- and 35-day-old piglets respectively. [Conclusion] The results highlight the suitability of both PCR assays in establishing the herd infection status of pigs in field conditions.

Immune Responses to the Attenuated Mycoplasma hyopneumoniae 168 Strain Vaccine by Intrapulmonic Immunization in Piglets

To investigate the immune responses to the attenuated Mycoplasma hyopneumoniae 168 strain vaccine, 8-15 d old piglets were immunized with M. hyopneurnoniae 168 strain vaccine by intrapulmonic route. And the specific IgG antibody in serum, lymphoproliferation, IFNT, and specific secretory IgA （SIgA） antibody in bronchoalveolar lavage fluid were detected on 30 and 60 d post-immunization （DPI）, respectively. On 60 DPI, all the pigs except for those in health control group were challenged with a field M. hyopneumoniae strain JS. Necropsy was performed on 30 d post-challenge （DPC）. The results showed that IFN7 and specific SIgA were stimulated on surface of respiratory tract after immunization. And peripheral blood mononuclear cells could also be proliferated about 1.81 and 2.12 fold on 30 and 60 DPI when stimulated by M. hyopneumoniae protein in vitro. However, no serum IgG antibody against M. hyopneumoniae was detected during the whole immune phage. After challenge, vaccinated pigs were observed with only very slight histological lesion in individual lobes. None of vaccinated pigs showed any clinical signs. While the unvaccinated pigs from challenge control group showed varying degrees of clinical sign and severe macroscopical lesion of mycoplasmal pneumonia of swine （MPS）. The result suggested that the attenuated M. hyopneumoniae 168 strain vaccine inoculated by intrapulmonic route could activate the systemic cellular immunity, the local mucosal immunity and IFNγ secretion in respiratory tract to against M. hyopneumoniae infection in piglets.

Standardization of a Real-time PCR System for Quantitative Detection of Mycoplasma hyopneumoniae

This study was conducted to develop a method for accurate quantification of Mycoplasma hyopneumoniae during vaccine production or experimental research. Primer and probe concentration that gave the highest ΔRn and the lowest Ct were selected to establish the real-time PCR system for the detection of M. hyopneumoniae. Template DNA of M. hyopneumoniae was extracted by boiling under different conditions and detected by real-time PCR to determine the optimal conditions for DNA extraction. Thereafter, intra-and inter-batch reproducibility tests were carried out using a standard plasmid to evaluate the stability of the PCR system. Subsequently, the effect of medium composition on the quantitative detection was evaluated. Finally, the correlation between real-time PCR and CCU method was explored. The optimal primer and probe concentration for real-time PCR were 0.4 and 0.2 μmol/L, respectively. The intra-and inter-batch coefficients of variation(CV) in Ct value of 10~4-10~9 copies/μl standard plasmid were <5%, indicating good reproducibility of the real-time PCR system. Following incubation in a boiling water bath for 10 min, M. hyopneumoniae samples can be used directly as a template in subsequent real-time PCR assays,and good intra-batch and inter-batch reproducibility was observed. The working concentration of KM2 medium should be less than the 1/10 of the concentration of the stock solution to minimize its influence on the quantitative detection. Spearman's correlation analysis revealed that the log of CCU and the log of DNA copy number had a significant positive relationship(r=0.797,P=0.000). Thus, the two methods can be used in combination in the quantitative detection of M. hyopneumoniae. In summary, a rapid, stable and accurate quantitative PCR system for detecting M. hyopneumoniae culture was established in this study, which provides a technical means for accurate quantification of M. hyopneumoniae in vaccine production and laboratory tests.

Presence of <i>Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae</i>in upper respiratory tract of swine in farms from Aguascalientes, Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms inAguascalientes,Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms);17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2;30% were positive for H. parasuis (93% of farms);23% of the samples to P. multocida (in 79% of farms);and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied;therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.

Establishment of a model of Mycoplasma hyopneumoniae infection using Bama miniature pigs

Mycoplasma hyopneumoniae(M.hyopneumoniae),is the primary aetiological agent of enzootic pneumonia leading to chronic respiratory disease prevalent worldwide.Conventional pigs are the only animals used for pathogenicity studies and vaccine evaluations of M.hyopneumoniae.Considering that the challenge animals have better genetic stability and a smaller body size to operate with,an alternative experimental animal model of M.hyopneumoniae infection with Bama miniature pigs was established.Nine seven-week-old snatch-farrowed,porcine colostrum-deprived(SF-pCD)Bama miniature pigs and nine conventional pigs were randomly divided into two infected groups(Bama miniatureinfected(BI)and conventional-infected groups(CI),BI and CI,n=6)and two control groups(Bama miniature control(BC)and conventional control(CC)groups,BC and CC,n=3).Every piglet was tracheally inoculated with 5×10^(8) CCU/mL containing 10%suspension of a stock of frozen lung homogenate from SF-pCD pigs infected with virulent strain JS or sterilized KM2 medium.Typical lung lesions appeared in all infected pigs after necropsy,and the mean gross lung lesions was 17.3 and 13.7 in groups of BI and CI.Serum IgG and nasal sIgA antibody titres were increased significantly.Cilia shedding and mucus staining increased greatly in JS-infected bronchi.Obvious reddish gross lesions and M.hyopneumoniae antigen were detected,especially apparently observed in group of BI.Moreover,DNA copies of M.hyopneumoniae from bronchoalveolar lavage fluid(BALF)of each JS-infected piglet reached more than 10^(8),and M.hyopneumoniae could be re-isolated from each infected BALF.These results indicate that Bama miniature pigs could be used as an alternative and more maneuverable experimental infection model for M.hyopneumoniae and display typical clinical and pathological features consistent with those in conventional pigs.

Secretory Expression of Mycoplasma hyopneu-moniae P97R1 Gene in Pichia pastoris and Primary Application of the Expression Product

[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.

猪肺炎支原体通过抑制SPLUNC1功能破坏呼吸道炎性反应平衡

【背景】猪肺炎支原体(Mycoplasmahyopneumoniae,Mhp)通过定植猪呼吸道黏膜层,破坏呼吸道炎性反应平衡,引起炎性损伤和持续性感染。短的上腭、肺及鼻咽上皮克隆1蛋白(short palate lung and nasal epithelial clone1,SPLUNC1)是呼吸道黏膜分泌的具有重要抗菌和抗炎功能的蛋白,被认为是呼吸道黏膜面对危险信号时的“信号传感器”。【目的】从Mhp和SPLUNC1相互作用入手,分析Mhp感染对SPLUNC1表达的影响以及SPLUNC1对Mhp引起的炎性反应的调控作用,揭示Mhp引起炎性损伤的新机制,为解决Mhp持续性感染问题提供参考。【方法】利用猪肺炎支原体对猪支气管上皮细胞(porcine bronchial epithelial cells,PBECs)感染模型和猪体感染模型,通过荧光定量PCR、间接免疫荧光和Western-blotting等方法,分别检测Mhp感染后对肺脏中SPLUNC1以及体外PBECs中SPLUNC1转录和表达的影响。克隆并扩增猪源SPLUNC1基因通过酶切鉴定,构建成功SPLUNC1真核和原核表达重组质粒pCDNA3.1-SPLUNC1以及pET28a-SPLUNC1。与此同时,设计靶向SPLUNC1的siRNA干扰片段。利用体外SPLUNC1蛋白孵育、体内呼吸道黏膜SPLUNC1抗体封闭,通过Mhp CCU50检测明确SPLUNC1对Mhp体内外生长的影响。在猪支气管上皮细胞中,通过过表达或siRNA干扰SPLUNC1基因,后感染Mhp,利用Western-blotting、间接免疫荧光以及酶联免疫吸附方法,明确SPLUNC1对Mhp的黏附作用、诱导炎性因子表达以及MAPK信号通路活化的影响。【结果】Mhp感染猪后可引起肺脏实变,主要组织病理表现为肺脏炎性损伤,同时显著上调肺泡灌洗液中趋化因子CXCL8和炎性因子TNFα和IL-1β分泌。Mhp体内外感染后,体内肺脏和体外猪支气管上皮细胞中SPLUNC1的转录和表达均显著下调。以上研究表明,Mhp可诱导肺脏炎性反应,抑制SPLUNC1表达。另一方面,体外PBECs中过表达SPLUNC1后,Mhp感染引起的CXCL8表达显著减少;siRNA干扰SPLUNC1后,Mhp感染引起的CXCL8表达显著增加,表明SPLUNC1负调控Mhp感染引起的CXCL8表达。基于Mhp感染入侵呼吸道过程,本研究进一步解析SPLUNC1负调控CXCL8表达的机制。结果表明:无论是SPLUNC1和Mhp体外孵育,还是SPLUNC1抗体体内封闭,Mhp的体内外生长均未受影响;SPLUNC1的过表达或siRNA干扰对Mhp体外黏附PBECs的能力也无显著影响;过表达SPLUNC1可抑制pERK和IκBα的激活,相反SPLUNC1 siRNA干扰后,可促进pERK和IκBα的激活。以上研究证明SPLUNC1不是通过调控Mhp的生长和黏附,而是通过负调控MAPK-ERK通路的活化来调控CXCL8的表达。【结论】SPLUNC1可通过MAPK-ERK信号通路来调控炎性因子的过度表达,维护宿主炎性平衡;与此同时,Mhp感染后可通过抑制SPLUNC1的表达来破坏宿主炎性反应的平衡调控,进而引起炎性损伤。本研究为解析Mhp感染损伤机制提供依据。

猪肺炎支原体LAP的结构预测及其抑制剂Bestatin对支原体生长的影响

本研究旨在以猪肺炎支原体为代表,研究支原体LAP酶活中心的高级结构及其被抑制后对支原生长的影响。亮氨酸氨肽酶(LAP)是一类广泛存在于各类生物的氨基酸代谢酶,有研究表明病原微生物的LAP酶活中心被抑制后可以直接影响微生物的代谢和生长。本研究拟以猪肺炎支原体(Mhp)LAP为模型,研究其理化特性和高级结构,探索LAP抑制剂乌苯美司(Bestatin)对支原体生长的影响。本研究首先用Clustal Omega进行序列同源性比较,分析LAP活性位点在不同支原体中的保守情况。然后采用GST融合标签表达猪肺炎支原体LAP蛋白,通过GST亲和层析和分子筛层析纯化蛋白,圆二色谱测定LAP的二级结构,用Alpha fold 2预测三级结构。最后用Bestatin与猪、鸡、羊等不同种属的支原体共培养来观察其影响。结果表明虽然总体序列同源性不高,但是LAP酶活中心的关键位点在常见支原体相对保守。猪肺炎支原体LAP可以通过GST标签进行可溶性表达。酶切GST标签后,分子筛层析显示LAP是六聚体,圆二色谱表明LAP具有正确的二级结构。预测的LAP三级结构显示其具有保守的底物结合关键位点。Bestatin对不同种属支原体的生长均产生抑制,且呈现剂量依赖性,但在不同菌株间存在差异,最低在6μg/mL的浓度可通过抑制氨肽酶的活性抑制猪肺炎支原体的生长。该研究为新一代抗菌药物的研发提供了新的思路。

关于我国种猪场猪肺炎支原体净化的思考

猪肺炎支原体是一种对生猪影响较大的细菌性病原体,往往可引起猪群发生慢性感染,且难以从猪群和猪场水平上清除,导致养猪业遭受巨大的经济损失。猪肺炎支原体净化在欧洲和北美已全面启动,且有成功案例,我国才刚刚启动。本文结合国外猪肺炎支原体净化方案的经验与我国生猪养殖特殊性的分析,提出分群体、分阶段的“三步法”净化建议,并对我国猪肺炎支原体的净化提出了展望。

猪肺炎支原体解螺旋酶RuvA的原核表达、多克隆抗体制备及活性鉴定

猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。

猪肺炎支原体P46-P65重组蛋白的表达及间接ELISA抗体检测方法的建立

为建立猪肺炎支原体(Mhp)血清学调查及免疫评估方法,本研究利用DNAStar生物学软件对Mhp的P46和P65进行抗原表位分析,利用重叠延伸PCR(SOE-PCR)获得P46-P65融合基因,构建重组表达质粒pETP46-P65,将其转化大肠杆菌BL21(DE3)感受态细胞,经诱导后获得了可溶性表达的重组P46(aa33~aa419)-P65(aa307~aa627)蛋白(rP46-P65)。以纯化的r P46-P65为包被抗原,经优化各反应条件后建立了检测Mhp抗体的间接ELISA方法。特异性试验结果显示所建立的方法与CSFV、FMDV、PEDV、PCV2、PRV、PRRSV和APP等阳性血清均无交叉反应。该方法可检测到最高稀释至6 400倍的Mhp阳性血清,批内和批间变异系数均小于5%。利用IDEXX试剂盒和本研究建立的方法同时检测298份临床血清样品,前者的检测阳性率为62.4%(186/298),后者的检测阳性率为73.8%(220/298),两者检测结果的总符合率为88.6%。IDEXX检测为阳性的血清,采用本研究建立的ELISA方法检测也均为阳性;而部分Mhp阳性猪经IDEXX试剂盒检测为阴性的血清,该ELISA方法检测结果却为阳性,其中79.4%(27/34)检测结果有差异的血清经Mhp颜色变化试验证明均为阳性,表明本研究建立的ELISA方法的敏感性明显高于IDEXX方法。本研究建立的检测猪Mhp抗体的间接ELISA方法与目前广泛使用的方法相比优势明显,为临床血清流行病学调查及血清抗体水平评估提供了可行方法。

猪肺炎支原体实时荧光定量PCR检测方法建立及应用

猪肺炎支原体(Mycoplasma hyopneumoniae)是一种严重危害养猪业的重要传染性病原,以猪肺炎支原体编码乳酸脱氢酶的P 36基因保守序列为基础,设计一套特异性引物和探针,并通过优化筛选反应条件,建立了一种Mhp的实时荧光定量RT-PCR检测方法。结果表明,最适引物浓度为10μmol/L,探针浓度为5μmol/L,以浓度为1×10^(2)~1×10^(6)copies/μL的标准品构建标准曲线相关系数为0.998,扩增效率可达96%。该方法能特异地检测猪肺炎支原体,与其他动物支原体及猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病病毒等病原无交叉反应;灵敏度是常规PCR的10倍,可达到10拷贝;该方法重复性较好,组内和组间变异系数均小于2%。在对30份临床样本的检测中,建立的实时荧光定量PCR检出率为63%(19/30),而常规PCR的检出率仅有30%(9/30)。结果表明,成功建立了猪肺炎支原体实时荧光定量PCR检测方法,可用于猪肺炎支原体的病原学检测和流行病学调查。

2022年中国南方部分地区猪肺炎支原体感染状况的调查与分析

猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)是猪地方性肺炎(EP)的原发性病原,可导致猪咳嗽、气喘、消瘦,并因猪的饲料转化率升高、日增重减少、出栏时间延长,从而使养殖成本大大增加,给养猪业造成重大经济损失。本研究旨在对中国南方部分地区猪肺炎支原体的感染状况进行调查与分析。从中国南方部分地区21个规模化种猪场定点采样,首先通过病原检测比较鼻拭子和喉拭子的采样效果,随后以简单随机抽样结合分层抽样确定各猪场样本量。共收集母猪喉拭子样品1478份,并将调查的猪群按胎次分为“0~1胎母猪”和“≥2胎母猪”,通过实时荧光定量PCR进行病原检测,通过多位点序列分型(MLST)结合保守的p36基因及非保守的p97和p146基因进行分子流行病学分析。检测结果表明,鼻拭子样品的检出率为16.67%,喉拭子样品的检出率为56.67%,且Ct值更低。猪肺炎支原体感染的场检出率为66.67%,但各场感染压力较小,检出率为1.39%~18.57%。其中“0~1胎母猪”检出率为10.05%;“≥2胎母猪”检出率为2.17%。分子流行病学结果显示,7个猪场的流行菌株均为同一序列类型(ST128),不同猪场间的流行菌株高度同源。对于猪肺炎支原体的病原学检测,喉拭子是比鼻拭子更灵敏且有效的活体样品类型,母猪胎次和生猪调运引种是规模化种猪场感染猪肺炎支原体的重要因素。本研究为猪肺炎支原体的防控和净化工作提供理论基础。

猪肺炎支原体Mhp-1株的分离鉴定及其全基因组测序分析

为了分离与鉴定引起的猪群疑似支原体性肺炎的病原体,本实验收集了20份病死猪肺脏病变组织,采用猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)特异性引物进行PCR鉴定、分析培养特性、颜色变化单位(CCU)测定、传代培养后扩增16S rRNA基因并测序分析等,并进一步采用BLAST分析分离菌16S rRNA基因序列的同源性,采用邻接法构建16S rRNA基因的系统进化树,对该分离株MLST分型、全基因组测序和毒力基因预测。结果显示,20份临床样品仅3份样品为Mhp单阳性,接种KM2液体培养基后仅1份生长良好,颜色规律性变黄;接种KM2固体培养基培养7 d后呈现特异性“煎蛋样”的菌落形态;瑞氏姬姆萨染色结果可见环状、点状、丝状、两级状等形状且大小不等的菌体形态,传12代后经特异性PCR检测结果为Mhp单阳性,表明该分离株可以稳定传代,且无Mhr污染;该分离株在KM2培养基上为1×10^(7)CCU/mL;16S rRNA基因序列比对结果显示该分离株与GenBank中登录的Mhp菌株ES-2(CP038641.1)同源性达100%。以上结果确定本研究分离出一株Mhp,并将其命名为Mhp-1。16S rRNA基因系统发育树分析结果显示,分离株Mhp-1与国外猪源Mhp分离株F7.2(KY264125.1)处于同一分支,亲缘关系最近;MLST分型结果显示Mhp-1分离株属于ST184型;全基因组测序获得序列大小为0.91 Mb,与已报道的Mhp基因组大小基本一致;毒力基因预测结果显示其存在EF-Tu、Lttp、P216、P102、P46、P97、PDH-B、Capsule、HlyA等毒力因子编码基因。本研究进一步丰富了Mhp的研究数据,为规模化猪场猪支原体性肺炎的防控提供参考数据。

猪圆环病毒2型、猪肺炎支原体和猪瘟疫苗不同免疫程序的免疫效果比较分析

为了评估不同免疫策略对猪场免疫效果和经济效益的影响,以确定最佳的疫苗免疫程序,本试验将处于产前1个月的四胎次健康三元母猪随机分为2个组,A组(联合免疫组)母猪于产前27 d联合免疫猪圆环病毒2型基因工程疫苗、猪肺炎支原体灭活疫苗和猪瘟活疫苗(圆柯欣+柯喘宁+稳柯健),其所产仔猪于21日龄联合免疫圆柯欣、柯喘宁和稳柯健;B组(对照组)母猪于产前34和27 d分别免疫稳柯健、猪圆环病毒疫苗和猪肺炎支原体疫苗二联疫苗(圆-支二联疫苗),其所产仔猪于14日龄免疫圆-支二联疫苗,28日龄免疫稳柯健。统计和分析免疫各组临床指标、生产性能和持续性抗体水平。结果显示,免疫疫苗后,2个组的母猪采食情况均正常;2个组的仔猪整体精神状态良好,哺乳情况正常,均未出现咳嗽和喘气等呼吸道疾病。在试验期间内,2个组的母猪的窝产健仔数和仔猪出生健仔均重并无差别。A组和B组的出生至23日龄死淘率、出生至50日龄死淘率分别为3.77%、5.11%和4.07%、5.43%,A组略优于B组。抗体监测结果显示,A组母猪的CSFV和PCV2抗体水平较B组无显著差异(P>0.05);A组仔猪的CSFV抗体阳性率与B组无明显差异(P>0.05),而PCV2抗体水平在免疫早期阶段(21日龄)明显高于B组(P<0.05)。结果表明,猪圆环病毒2型、猪肺炎支原体和猪瘟疫苗可以联合免疫,且免疫后相互之间不干扰。该联合免疫策略在确保免疫效果的同时,简化了疫苗免疫程序,为确定最佳的疫苗免疫程序提供了有价值的临床应用参考。