Actinobacillus pleuropneumoniae, a significant respiratory pig pathogen, is causing substantial losses in the global swine industry. The resistance spectrum of A. pleuropneumoniae is expanding, and multidrug resistance is a severe issue. Horizontal gene transfer (HGT) plays a crucial role in the development of the bacterial genome by facilitating the dissemination of resistance determinants. However, the horizontal transfer of resistance genes via A. pleuropneumoniae-derived outer membrane vesicles (OMVs) has not been previously reported. In this study, we used Illumina NovaSeq and PacBio SequeI sequencing platforms to determine the whole genome sequence of A. pleuropneumoniae GD2107, a multidrug-resistant (MDR) isolate from China. We detected a plasmid in the isolate named pGD2107-1; the plasmid was 5,027 bp in size with 7 putative open reading frames (ORF) and included the floR resistance genes. The carriage of resistance genes in A. pleuropneumoniae OMVs was identified using a polymerase chain reaction (PCR) assay, and then we thoroughly evaluated the influence of OMVs on the horizontal transfer of drug-resistant plasmids. The transfer of the plasmid to recipient bacteria via OMVs was confirmed by PCR. In growth competition experiments, all recipients carrying the pGD2107-1 plasmid exhibited a fitness cost compared to the corresponding original recipients. This study revealed that OMVs could mediate interspecific horizontal transfer of the resistance plasmid pGD2107-1 into Escherichia coli recipient strains and significantly enhance the resistance of the transformants. In summary, A. pleuropneumoniae-OMVs play the pivotal role of vectors for dissemination of the floR gene spread and may contribute to more antimicrobial resistance gene transfer in other Enterobacteriaceae.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that clinically causes severe swine and human infections (such as meningitis, endocarditis, and septicemia). In order to cause widespread diseases in different organs, S. suis 2 must colonize the host, break the blood barrier, and cause exaggerated inflammation. In the last few years, most studies have focused on a single virulence factor and its influences on the host. Membrane vesicles (MVs) can be actively secreted into the extracellular environment contributing to bacteria-host interactions. Gram-negative bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide (LPS), causing host cell pyroptosis. However, little is known about the effect of the MVs from S. suis 2 (Gram-positive bacteria without LPS) on cell pyroptosis. Thus, we investigated the molecular mechanism by which S. suis 2 MVs participate in endothelial cell pyroptosis. In this study, we used proteomics, electron scanning microscopy, fluorescence microscope, Western blotting, and bioassays, to investigate the MVs secreted by S. suis 2. First, we demonstrated that S. suis 2 secreted MVs with an average diameter of 72.04 nm, and 200 proteins in MVs were identified. Then, we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis. The S. suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway, resulting in cell pyroptosis, but it did not activate the Caspase-4/-5 pathway. More importantly, endothelial cells produce large amounts of reactive oxygen species (ROS) and lost their mitochondrial membrane potential under induction by S. suis 2 MVs. The results in this study suggest for the first time that MVs from S. suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage, which produced mtDNA and ROS under induction, leading to the pyroptosis of endothelial cells.

[Objective]The study was carried out to understand the genetic evolution of Porcine Circovirus Type 2(PCV2)strains on a swine farm in Guangdong Province,and enrich molecular epidemiological data of PCV2,in order to provide reference for the selection and development of local PCV2 vaccine candidate strains.[Method]Samples suspected of PCV2 infection were detected by using qPCR methods.A PCV2-positive isolate with a high viral load was found,named GD222858.Genome-wide molecular cloning and genetic evolution analysis were performed with PCR methods.MegAlign software was used to compare the amino acid sequences encoded by the ORF1 and ORF2 genes of the strain with the PCV2 isotype reference strain,and the similarity of the amino acid sequences was analyzed.The DNAStar was used to predicte the Cap protein secondary structure and B-cell epitope of this strain and compare it with the Cap protein antigen index of four vaccine strains DBN-SX07-2(HM641752),LG(HM038034),SH(HM038027)and ZJ(AY686764).[Result]Sequencing results showed that the genome length of GD222858 strain was 1 767 bp.The genetic evolution analysis indicated that the strain belonged to the PCV2d subtype.The nucleotide similarity with 82 reference strains at home and abroad ranged from 91.4%to 99.6%.It was closest to the Vietnamese strain Han8(GenBank accession No.:JQ181600).Multiple specific mutation sites F70Y,F77L,W202R and N256S were found at the Rep protein encoded by ORF1.The Cap protein encoded by ORF2 was relatively conserved.Protean predicted that the potential B cell epitopes presented at amino acid positions 5-18,24-25,39-41,48-49,57-65,99,101,112-114,139-140,145-150,162-165,175-181,188-189,205-211 and 227-232 of Cap proteins.The Cap protein antigen index of the GD222858 strain was different from that of the four vaccine strains,and the antigen index at the amino acids 45-57,124-132 and 223-233 was significantly higher than that of the other four vaccine strains,and the difference with the LG vaccine strain(HM038034)was greatest.[Conclusion]The reason for the infection of GD222858 strain in pig herds may be due to specific mutations in multiple sites of the Rep protein and improper selection of vaccine strains.

为掌握紫金县高致病性禽流感、新城疫、非洲猪瘟、高致病性猪蓝耳病、口蹄疫、猪瘟、猪伪狂犬病、布鲁氏菌病、小反刍兽疫等动物重要疫病流行及疫苗免疫情况,对2021年紫金县采集的4 978份畜禽样品进行了病原学及血清学检测及监测.检测结果显示,2021年紫金县重要动物疫病免疫抗体水平均较高,未检测出重要动物疫病病原,这表明紫金县重要动物疫病免疫效果及防控效果均较好.

为了建立一种高效、灵敏的胸膜肺炎放线杆菌(APP)检测方法,本研究针对APP的血清型保守基因-毒素4(APxⅣ)序列合成特异性引物,通过优化引物浓度、退火温度等建立了APP纳米PCR检测方法.敏感性试验结果显示,该纳米PCR对APP的检测下限为1×102 CFU/mL,其灵敏度是普通PCR的10倍;特异性结果显示,该方法仅从APP核酸样品可检测到约417 bp的特异性目的条带,而对猪链球菌、副猪嗜血杆菌、大肠杆菌、沙门菌、金色葡萄球菌等其他病原的检测结果均为阴性;应用本研究建立的纳米PCR技术检测了41份临床疑似APP感染病料,结果显示常规PCR和纳米PCR检测APP阳性率分别为17.07％(7/41)和19.51％(8/41).以上结果表明,本研究成功建立的APP纳米PCR检测方法敏感性高、特异性好,可用于APP感染的临床诊断及流行病学调查.

In this study,a highly sensitive and specific detection method was established based on the B646L（P72） protein gene of African swine fever virus （ASFV）.The nested primers and probes of LNA-OSN-PCR （One Step Nested-Locked Nucleic Acid real-time PCR） were designed with Oligo 7 software;external primers were modified with locked nucleic acids.The reaction system and conditions of LNA-OSN-PCR were established and optimized,and the standard curve of LNA-OSN-PCR was established.Then,the sensitivity,specificity and repeatability of LNA-OSN-PCR were tested.A total of 96 clinical suspected diseased samples were detected by LNA-OSN-PCR and compared with the samples detected by conventional fluorescent PCR method.The reaction conditions and system of LNA-OSN-PCR were established and optimized,and the standard curve of LNA-OSN-PCR had good linearity （R~2=0.9945）.The LNA-OSN-PCR had a minimum detection limit of 3×10 -1 copies/μL,with a coefficient of variation of less than 3%and no cross-reaction with other viral or bacterial nucleic acids.Compared with the qPCR method recommended by OIE,the detection sensitivity of the LNA-OSN-PCR method was increased by 10-100 times.Among the 96 samples tested by LNA-OSN-PCR,55 were positive and 41 were negative,with a higher positive detection rate than traditional qPCR and a better typical amplification curve.By combining the one-step nested PCR and lock-in nucleic acid modification,the LNA-OSN-PCR method for ASFV was established to provide a highly sensitive,specific and economical method for ASFV detection.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that clinically causes severe swine and human infections (such as meningitis, endocarditis, and septicemia). In order to cause widespread diseases in different organs, S. suis 2 must colonize the host, break the blood barrier, and cause exaggerated inflammation. In the last few years, most studies have focused on a single virulence factor and its influences on the host. Membrane vesicles (MVs) can be actively secreted into the extracellular environment contributing to bacteria-host interactions. Gram-negative bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide (LPS), causing host cell pyroptosis. However, little is known about the effect of the MVs from S. suis 2 (Gram-positive bacteria without LPS) on cell pyroptosis. Thus, we investigated the molecular mechanism by which S. suis 2 MVs participate in endothelial cell pyroptosis. In this study, we used proteomics, electron scanning microscopy, fluorescence microscope, western blotting, and bioassays, to investigate the MVs secreted by S. suis 2. First, we demonstrated that S. suis 2 secreted MVs with an average diameter of 72.04 nm, and 200 proteins in MVs were identified. Then, we showed that MVs were transported to cells via and dynamin-dependent endocytosis. The S. suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway, resulting in cell pyroptosis, but it did not activate the caspase-4/-5 pathway. More importantly, endothelial cells produce large amounts of reactive oxygen species (ROS) and lost their mitochondrial membrane potential under induction by S. suis 2 MVs. The results in this study suggest for the first time that MVs from S. suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/caspase-1/GSDMD pathway by mitochondrial damage, which under induction produced mtDNA and ROS, leading to the pyroptosis of endothelial cells.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/μL, 6.48 × 105 copies/μL, 8.54 × 105 copies/μL and 7.79 × 106 copies/μL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

[目的]了解广东省部分规模化猪场引起猪传染性胸膜肺炎(PCP)的病原菌猪传染性胸膜肺炎放线杆菌(APP)的耐药表型和耐药基因携带情况,并分析比较其相关性,为有效防控该病提供理论依据.[方法]将2019-2021年从广东省不同地区规模化猪场采集的疑似猪传染性胸膜肺炎病死猪病料通过病原分离培养、革兰氏染色、生化试验、PCR扩增等方法进行病原菌分离鉴定和测序分析,随后对分离株进行药敏试验并通过PCR检测其耐药基因,确定其耐药表型和耐药基因的携带率,分析比较两者的一致性.[结果]分离菌需在含有血清和NAD的培养板中生长;革兰氏染色结果显示分离菌为红色,可判定为革兰氏阴性细菌,通过生化试验、PCR结果及测序分析确定分离到20株APP,且没有表现出明显的地域特性.所有菌株均呈现多重耐药且约50％菌株呈8重及以上耐药;其中对四环素类、磺胺类、氯霉素类和大环内酯类药物耐药率较高,分别为77.5％、65.0％、55.0％和48.8％;进一步分析发现,分离菌主要对四环素、磺胺异噁唑、氟苯尼考和林可霉素等抗菌药物耐药,而对头孢唑啉(先锋V)和阿奇霉素敏感.23种主要耐药基因中共检测到blaCMY、aphi(2")-Ⅰ b、sul1、sul2、su13、tetA、tetB、tetM、tetO、tetR和floR 这 11 种耐药基因,携带率分别为 85％、85％、50％、60％、75％、30％、100％、85％、100％、70％和 80％;未检测到喹诺酮类、大环内酯类和林可胺类相关耐药基因.[结论]从广东省猪群中分离到的APP表现出广泛耐药性,且为多重耐药,耐药基因与耐药表型基本一致,表明耐药基因的携带是细菌对抗菌药物产生耐药的主要原因之一,但也可能存在其他未检测的耐药基因,或存在新的耐药机制.

[目的]通过对多重耐药胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)GD2107株进行全基因组测序及生物信息学分析,丰富APP基因组数据库信息;构建基于ApxⅣ基因的系统进化树,分析该菌株的进化关系,为探索APP致病机制和临床防控猪传染性胸膜肺炎(porcine contagious pleuropneumia,PCP)提供参考.[方法]通过药敏试验测定分离菌株的耐药谱;采用全基因组测序技术(whole genome sequencing,WGS)对细菌DNA进行全基因组测序,分别利用Illumina NovaSeq、PacBio SequeⅠ测序平台对全基因组测序结果进行基因功能注释及生物信息学分析(包括基因组基本信息、功能元件分析及亚系统分析等);基于ApxⅣ基因构建系统进化树.[结果]药敏试验结果显示,GD2107菌株对青霉素、头孢拉定(先锋VI)、卡那霉素等14种抗菌药均耐药.对GD2107株全基因组测序得到1条大小为2 271 987 bp的环状染色体(GC含量为41.21％)和2个大小分别为5 027和3 497 bp的环状质粒,共预测到2 290个编码基因,包含19个rRNA(7个5S rRNA、6个16S rRNA、6个23S rRNA)、21个tRNA基因、20个ncRNA;23个基因岛、4个原噬菌体和2组CRISPR相关序列;分别有2 112、1 549和1 866个基因在COG、KEGG和GO数据库中得到注释,且相关蛋白集中分布于APP的代谢过程;另外在毒力因子(VFDB)和耐药因子(CARD)数据库中还注释到48个毒力基因和22个耐药基因(仅floR基因位于质粒上).绘制该菌株的全基因组圈图,将基因组信息提交至NCBI,获得染色体GenBank登录号为CP097377,质粒登录号分别为CP097378和CP097379.系统进化树分析发现,该菌株与来自中国的APP菌株(CP063424.1)进化关系最近.[结论]本研究完成了对多重耐药菌株GD2107的全基因组测序与生物信息学分析,全面认识了该菌株基因组的结构和功能并探究了耐药和致病机制中的相关基因,进化关系显示该菌株具有一定的地域流行性,为预防PCP的流行和探索APP的致病机制提供了参考.

为了确定广东省某规模化猪场病死猪的发病原因,走访猪场的了解状况,解剖并采集发病猪只的心脏、肺脏等病变组织进行实验室检测鉴定,并对分离到的致病菌进行血清型鉴定和药敏试验分析.结果显示,该猪场感染了猪胸膜肺炎放线杆菌(Actinobacillus pleuropneu?moniae,APP)7型和猪链球菌(Streptococcus suis,SS)2型.其中夏季高温高湿的环境应激是细菌病的主要诱发因素,并根据确诊结果和猪场整体环境情况提出相应的防治建议.

根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因核苷酸序列设计特异性引物和锁核酸(locked nucleic acid,LNA)-TaqMan探针,建立了基于P72基因的LNA-TaqMan探针的ASFV荧光定量PCR方法.结果显示,所建立的LNA-TaqMan探针荧光定量PCR方法具有较高的灵敏度,最低检测限为3.9拷贝/μL,且与猪瘟病毒、猪繁殖与呼吸综合征病毒及猪圆环病毒2型等多种病原不存在交叉反应;该方法的重复性良好,批内和批间变异系数均小于1％;56份临床样品的检测结果与O1E推荐的qPCR方法检测结果一致,符合率为100％.结果表明,本试验所建立的ASFV LNA-TaqMan探针荧光定量PCR方法敏感性、特异性和重复性良好,为ASFV检测提供了一种新的技术选择.

猪伪狂犬病是一种由伪狂犬病病毒(Pseudorabies virus,PRV)感染引起的急性高度接触性传染病,可造成不同日龄的猪发病,给养猪业造成了严重的经济损失.预防、控制甚至消灭猪伪狂犬病的主要措施之一是疫苗免疫接种.通过基因重组技术对PRV关键毒力基因进行改造,敲除毒力基因或插入免疫增强基因,是降低PRV毒力或提高病毒免疫保护效果的有效方法之一,也是研制安全高效PRV基因工程疫苗的重要手段.从基因同源重组、Cre/lox P位点特异性重组、细菌人工染色体和CRISPR基因编辑等不同基因重组技术应用方面对猪PRV基因工程疫苗的研制进行综述,为PRV重组疫苗的进一步深入研究提供参考.

本研究应用血清抗性试验,检测了从仔猪不同部位分离的66株副猪嗜血杆菌的毒力,对比菌株对仔猪、兔、豚鼠的血清敏感性差异.试验结果显示:66株菌株中,35株是高抗血清的强毒力菌株,7株是中等抗血清的中等毒力菌株,24株是对血清敏感的无毒力菌株.说明不同分离部位的副猪嗜血杆菌对血清分别呈现血清敏感性和血清抗性,在致病力方面也存在显著差异.菌株对兔、豚鼠和猪血清敏感性相同,特异性抗体对副猪嗜血杆菌并没有更高的杀伤力.吖啶黄凝集试验显示,菌株对血清的敏感性水平和它的表面结构没有直接的联系.