Mycoplasma gallisepticum (MG) is a major avian respiratory pathogen responsible for chronic respiratory disease and substantial economic losses in the poultry industry worldwide. Live attenuated vaccines, including strains F, ts-11, and 6/85, are widely used for MG control; however, current diagnostic methods are unable to reliably differentiate wild-type MG infections from vaccine strain persistence or replication, complicating surveillance and control programs in vaccinated flocks. In this study, a rapid and discriminatory multiplex TaqMan real-time quantitative PCR (qPCR) assay was developed to simultaneously detect MG and differentiate wild-type strains from the three major commercial live vaccine strains (F, ts-11, and 6/85) in a single reaction. Strain- and species-specific target regions were identified through comparative genomic analysis of 19 MG genomes, and corresponding primers and probes were designed and optimized. Under optimal conditions, the assay exhibited excellent linearity (R2 = 0.995-0.998), amplification efficiencies ranging from 85.16% to 96.40%, and a limit of detection of 1 × 102 copies/μL for all targets, demonstrating a 10-fold higher sensitivity than conventional PCR. No cross-reactivity was observed with other common avian pathogens, including NDV, AIV, IBV, FAdV, ILTV, and MS. Application of the assay to clinical poultry samples demonstrated its ability to reliably distinguish vaccine strains from wild-type MG. Using this method, the prevalence of wild-type MG was investigated in 2,564 poultry samples collected in the Guangxi region. The results showed that vaccination was associated with inhibition of wild-type MG infection within flocks, whereas unvaccinated flocks exhibited a wild-type MG infection rate of 58.5% (416/711). In conclusion, this multiplex qPCR assay provides a sensitive and specific tool for differentiating wild-type MG from vaccine strains and may support routine diagnosis and improved control of MG in poultry production systems.

The N1L gene is a well-characterized virulence factor in the poxvirus family; however, its functional role in Goatpox virus (GTPV) remains poorly understood. To elucidate the biological significance of the GTPV N1L gene (gN1L), we constructed three recombinant vaccinia virus Tiantan strain (rVVT) using homologous recombination: rVVT-ΔvN1L (deletion of VVT N1L), rVVT-vN1Lr (insertion of enhanced green fluorescent protein, EGFP), and rVVT-gN1L (substitution with gN1L). The biological properties of these recombinant strains were systematically compared with those of wild-type VVT to evaluate the functional role of gN1L. Bioinformatics analysis revealed that the gN1L-encoded protein shares 26.80% homology and 45.10% similarity with the VVT N1L (vN1L)-encoded protein. Notably, the gN1 protein was predicted to be structurally stable, whereas the vN1 protein was classified as unstable. Growth curve assays demonstrated that gN1L significantly enhances VVT replication in BHK-21, HeLa, and PK-15 cells. RNA-seq analysis further suggested that this enhancement is potentially mediated through the PI3K/AKT signaling pathway. In vitro and in vivo virulence assays indicated that gN1L increases VVT virulence by up to 133-fold, representing a 7.5-fold greater effect compared to vN1L. Additionally, viral load measurements in host tissues revealed that gN1L facilitates VVT traversal across the blood–brain barrier by enhancing its ability to infect glial and endothelial cells. Collectively, these findings provide novel insights into the functional role of gN1L and offer valuable implications for the development of safer attenuated vaccines against GTPV.

IntroductionHuman papillomaviruses (HPVs) are well-known for causing both benign and malignant epithelial growths in humans, but their occurrence in non-human species is rarely reported. Expanding the understanding of HPV’s host range is essential for assessing its ecological and public health implications.MethodsWe investigated serum samples from dogs collected in Guangxi, China, between 2014 and 2020. PCR screening was performed to detect HPV DNA, followed by sequencing and phylogenetic analysis of the positive amplicons.ResultsHPV DNA was unexpectedly detected in 2 out of 1,226 canine serum samples, yielding a detection rate of 0.16%. Phylogenetic analysis revealed that the sequences clustered with alpha2-HPV78 (GX-70-related) and alpha2-HPV94 (GX-47-related), respectively. Interestingly, both sequences displayed multiple amino acid variations in viral proteins. However, virus isolation was not achieved.DiscussionThe detection of HPV nucleic acids in canine serum suggests a potential presence of HPV in canine hosts. These findings provide new insights into the possible host range of HPV, underscoring the need for further research to assess the virus’s infectivity, transmission dynamics, and implications for both animal and human health.

BACKGROUND:Canine circovirus (CanineCV), a non-enveloped virus with a circular DNA genome, has been identified in various avian and mammalian species, including domestic and wild canids. This study aimed to comprehensively analyze the prevalence of CanineCV across diverse animal species in 11 provinces of China. RESULTS:A total of 1,666 serum samples were collected, revealing a 5.82% prevalence of CanineCV in dogs, with the highest rates being observed in southern and eastern China. Phylogenetic analysis of 266 global CanineCV genomes sourced from the NCBI identified six distinct genotypes, elucidating the complex dynamics of their evolution. Evidence suggested a potential bat origin for CanineCV, with positive selection and high rates of evolution being observed. Recombination analysis revealed dynamic genetic exchange, highlighting the intricate nature of CanineCV evolution. Mutational analysis identified key amino acid substitutions likely to influence the virus's adaptation. Additionally, glycosylation, palmitoylation, and SUMOylation sites were predicted, shedding light on crucial functional properties of the virus. CONCLUSIONS:This study provides a global perspective on the origin, genetic diversity, and evolutionary dynamics of CanineCV. Understanding these factors is crucial for elucidating its epidemiology and potential health risks.

Sus scrofa papillomatosis (SsP) is a tumour caused by Sus scrofa papillomaviruses (SsPVs). To investigate the presence of SsPVs in China, 354 domestic pig skin samples collected from Guangxi Province were examined for SsPV DNA by PCR. Three SsPV1s (GX12, GX14, and GX18) were identified with a prevalence of 0.847% (3/354). Sequence analysis showed that L1 of SsPV1/GX12 and SsPV1/GX14 had 99.7% and 99.6% nucleotide identify with the reference SsPV1a, respectively. Phylogenetic and evolutionary analyses showed that SsPV1/GX12 and SsPV1/14 clustered into SsPV1a and that SsPV1/GX18 clustered into SsPV1b. Compared with other SsPV L1 and L2 proteins, we found that the SsPV1/GX18 and SsPV1b strains shared the same unique substitutions, and SsPV1/GX12, SsPV1/GX14, and SsPV1a shared almost identical amino acid sequences. This study reports the first detection of SsPV DNA in China based on whole genome information and provides a scientific basis for the development of SsPV pathogenic biology, epidemiology, and prevention, as well as control technology research.

为了解广西部分地区(玉林市、大新县、那坡县、东兴市、靖西市和百色市)猪群中猪链球菌(Streptococcus suis,SS)毒力基因的流行情况和毒力水平,试验采用PCR方法检测分离到的201株(经鉴定猪链球菌2&1/2型24株,猪链球菌7型2株,猪链球菌9型15株,一共41株)猪链球菌的毒力基因orf2、sly、ef和mrp,并通过斑马鱼模型筛选毒力较强的菌株,测定菌株的半数致死剂量(LD50).结果表明:从广西部分地区分离的201株猪链球菌的4种毒力基因携带率从高到低依次为orf2(85.6％,172/201)＞sly(55.2％,111/201)＞ef(33.8％,68/201)＞mrp(26.4％,53/201);6 个市县主要流行的毒力基因不同,玉林市、大新县、那坡县、东兴市、靖西市和百色市分离的猪链球菌携带最多的毒力基因分别为 orf2(100％)、orf2(89.2％)、orf2(91.5％)、ef(44.4％)、orf2(86.7％)、orf2(40.0％);经鉴定分型的41株猪链球菌中,有7株(17.1％,7/41)同时携带了 4种毒力基因,有11株(26.8％,11/41)同时携带了 3种毒力基因,有17株(41.5％,17/41)同时携带了 2种毒力基因;斑马鱼模型筛选出7株致病力较强猪链球菌,LD50 为4.2×107～3.8×108 cfu/mL,且猪链球菌2型菌株毒力和猪链球菌9型菌株毒力差别不大.说明广西部分地区分离的猪链球菌毒力基因携带率较高,各地区流行的毒力基因多样,对斑马鱼有明显的致病性,但毒力不强,未来应当更加关注猪链球菌9型的流行情况和毒力水平.

为探究广西部分地区猪群中猪链球菌(Streptococcus suis)的流行情况及耐药性,采集广西6个市县共527份猪组织及鼻腔拭子样品,通过选择培养基对样品中的猪链球菌进行分离和PCR鉴定,共分离到猪链球菌201株(38.1％).其中大新县和那坡县猪链球菌分离率最高,分别为64.0％(64/100)和65.4％(70/107),而靖西市截获的越南走私猪中猪链球菌分离率达29.3％(44/150).对分离到的猪链球菌进行血清型分型鉴定,结果显示201株猪链球菌血清型中2(1/2)型24株(11.9％),7型2株(1.0％),9型15株(7.5％),大新县、那坡县和东兴市主要流行2(1/2)型,靖西市的越南走私猪样品主要流行9型.采用纸片扩散法,测定41株广西猪链球菌分离菌株对临床上常见的34种9类抗生素的耐药性,其中有1株猪链球菌只对2类抗生素中度敏感,对7类抗生素不敏感;有12株(29.3％)细菌多重耐药;大多数猪链球菌(65.9％)对大环内酯类不敏感,对万古霉素(87.8％)、硝基呋喃(87.8％)、氟喹诺酮类(82.9％)敏感.由此可见广西地区猪链球菌分离株有较强的耐药性.

为了评价口蹄疫(FMD)O型、A型二价3B表位缺失灭活疫苗在奶水牛临床应用的安全性及免疫效果,本试验选择90～100日龄健康奶水牛犊牛24头,平均分为2个组,试验组接种表位缺失疫苗,在首免后30 d,二免后180 d加强免疫,对照组按照相同的免疫程序接种常规疫苗;在不同时间点采集血清进行口蹄疫O型、A型抗体效价和非结构蛋白3ABC抗体检测,采集食道-咽分泌液(OP)进行口蹄疫病原学检测.结果显示:在抗体平均滴度方面,试验组抗体比对照组高1.4～2.8个滴度,在9个免疫后时间点试验组O型抗体平均滴度全部显著(P＜0.05)甚至极显著(P＜0.01)高于对照组,7个免疫后时间点试验组A型抗体平均滴度显著(P＜0.05)甚至极显著(P＜0.01)高于对照组;试验组抗体合格率比对照组高出25％～41％;口蹄疫非结构蛋白3ABC抗体标记疫苗免疫动物均为阴性.综合分析表明,口蹄疫O型、A型二价3B表位缺失灭活疫苗具有良好的免疫保护效果,在感染抗体水平上也能保证非结构蛋白抗体阴性.本试验为口蹄疫净化和口蹄疫无疫小区、无疫区建设探索可行的方法.

2020年11月,广西河池市发生1起流浪犬咬伤人和牛事件,通过现场流行病学调查和实验室检测等判定为一起狂犬病疫情.将伤者送医并接受暴露后处置,同时对疫点的受伤牛、污染场地和其他犬只进行紧急处置.后续跟踪调查发现,伤者伤口已愈合,受伤牛发病后被扑杀且无害化处理;该地区犬和其他动物没有发现异常,也无狂犬病继发病例报告.本次突发公共卫生事件处置及时、诊断准确、防控措施有效,为该地区狂犬病防控工作提供参考.

2021年6月,广西某县一羊场检出布鲁氏菌阳性,怀疑当地暴发了羊布鲁氏菌病疫情.为确定病因,控制疫情,减少损失,防止类似事件再次发生,采用现场访谈、实地调查、问卷调查、采样检测等方式开展了流行病学调查.调查发现:此次疫情涉及该县2个乡镇3个行政村10个养殖场户;这10个阳性场户间有流行病学关联,存在混牧、公羊共用和销售调运羊只等情况.调查认为,违规引种而引进感染动物,不按规定对调入动物实施隔离饲养,养殖户生物安全意识差,是本次布鲁氏菌病疫情发生的主要原因.本次疫情警示,相关防疫部门应加强防疫知识及相关法规的宣传教育,提高养殖场户的生物安全意识和依法落实防疫主体责任意识,加强隔离饲养,避免混牧和公羊共用,依法规范引种、调运动物等行为.

牛结核病是一种由牛型结核分枝杆菌引起的慢性人兽共患传染病,该病一年四季均可发生,被我国列为二类动物疫病.2020年,桂平市某奶牛场发生疑似牛结核病,经皮内变态反应检测出阳性样品16份.采集这16份皮内变态反应阳性牛的抗凝血样采用ELISA方法进行牛结核病γ-干扰素检测,结果诊断出8头牛结核病阳性奶牛.感染奶牛由桂平市动物疫病预防控制中心进行扑杀处理.据此建议,养殖场应该做好检疫和生物安全措施,防止该病的发生,在监测环节中可先用皮内变态反应检疫牛群,对于阳性牛再结合γ-干扰素ELISA方法进行综合判断,将更有利于牛结核病的检测和净化.

为筛选可靠的牛结节性皮肤病免疫抗体评价方法,以已知牛结节性皮肤病感染牛血清17份、羊痘疫苗免疫牛/羊血清77份和阴性牛血清20份作为样品盘,以病毒中和试验结果作为金标准,通过Kappa一致性和诊断效能评价,分别对市售的4种牛结节性皮肤病/羊痘抗体ELISA检测试剂盒进行分析比对.结果显示:4种ELISA检测试剂盒特异性为87.5％～94.34％,敏感性均不足80％,其中2种试剂盒与病毒中和试验的符合率超过80％,一致性为高度.结果表明,在临床应用中应根据检测目的选用可靠的试剂盒.本研究为科学有效防控牛结节性皮肤病提供了技术支持.

牛结核病是由牛分枝杆菌引起的一种人兽共患慢性传染病,可通过病畜传播给人类或其他动物.临床上牛结核病以肺结核最为常见,但也可发生乳腺结核和肠结核等,健康牛可通过被污染的空气、饲料和饮水等途经呼吸道和消化道感染.此外,牛结核病是人结核的重要传染源之一,在发展中国家仍然是一个严重的公共卫生问题[1].牛结核病治疗难度大、临床症状不典型,净化工作相对困难.

为实现痘苗病毒的快速检测,根据痘苗病毒TA27L基因设计引物,经各项条件优化后,建立痘苗病毒SYBR Green Ⅰ荧光定量PCR检测方法,同时将PCR扩增的TA27L基因克隆至pMD18-T载体,将测序正确的重组质粒作为标准品进行敏感性、特异性和重复性试验.结果 显示,所建立方法的Ct值与标准品在7.58×109～7.58×102 copies/μL范围内呈良好线性关系,R2为0.997,斜率为-3.175,检测下限为75.8copies且具有良好的特异性.临床样本检测结果表明该方法较普通PCR方法的检出率更高.结果 表明,建立的痘苗病毒SYBR Green Ⅰ荧光定量PCR检测方法为痘苗病毒及其相关基因重组毒株的生物学特性研究提供了必要的技术手段.

根据GenBank上已发表的PCV3毒株序列设计1对检测引物和2对扩增全长引物.应用检测引物对广西地区的犬血清样品进行PCR检测,应用扩增全长引物对检测阳性样品进行PCR扩增、克隆和测序,并对其全基因序列进行分析,绘制遗传进化树.结果 显示,广西地区147份犬血清样品中,阳性样品为36份,感染率为24.3％.本试验成功扩增1株2 000 bp的PCV3毒株的全基因核苷酸序列,并命名为PCV3/Guangxi-5.将PCV3/Guangxi-5序列与NCBI公布的PCV3的参考序列进行同源性比对,结果显示PCV3/Guangxi-5与参考序列的全基因序列同源性为98.9％～100.0％,其中与DE27.16同源性最低,与PCV3/CN/Chongqing-148/2016同源性最高.应用MEGA7.0对PCV3进行ORF2基因的氨基酸序列进行比对发现,第24位至第27位氨基酸存在6种形式,分别为VRRK、ARRR、ARKR、LRRK、VRRR和ARRK.利用MEGA7.0软件中Maximum Likelihood (ML)法p-distance模式构建基于ORF2基因和PCV3全基因的系统发育分析表明,PCV3可分为PCV3a和PCV3b等2种基因型,本试验获得的PCV3/Guangxi-5为PCV3a亚型,其ORF2基因与PCV3/CN/Liaoning-23/2016亲缘关系最近,与NWHEB21、毒株亲缘关系相距最远;其全基因组与CN/Jiangxi-62/2016毒株亲缘关系最近,与CNFJ-1毒株亲缘性最远.结果 表明,广西地区犬群普遍存在感染PCV3的情况,理论上丰富了广西地区PCV3的流行病学资料,为PCV3的防治措施的制定和流行病学研究提供一定的参考依据.

从试剂成本、人工成本、奶牛产奶量等方面,对采用皮内变态反应和γ-干扰素ELISA检测方法检测奶牛场牛结核病对经济效益的影响进行对比.结果 表明:在测试期(11 d)内,采用牛结核病γ-干扰素ELISA方法较运用皮内变态反应方法,每头奶牛产生的经济效益高374.0元(按广西当前鲜奶收购价计算).对奶牛场进行牛结核病检测时,选择γ-干扰素ELISA方法更利于保障经济效益的最大化.

为填补国内外水禽源痘病毒培养特性和理化特性研究方面的空白,对国内检测并鉴定的鹅源禽痘病毒,用鸡胚进行分离培养和病毒理化特性研究.将鹅皮肤痘疹样本研磨后,接种SPF鸡胚进行连续传代培养.病理检查和PCR检测证实,病毒可在鸡胚中增殖,且经连续5轮传代后,鸡胚病变及病毒滴度趋于稳定.鸡胚中可以观察到绒毛尿囊膜水肿增厚及白色痘斑等禽痘特征性病变.对采集病变的绒毛尿囊膜进行电子显微镜观察,可见病毒包涵体和典型禽痘病毒粒子.利用建立的鸡胚培养体系,对该病毒理化特性进行鉴定,发现病毒对热(55℃)、酸(pH3)、碱(pH11)、胰蛋白酶、乙醚和氯仿敏感.本研究首次建立了鹅源禽痘病毒鸡胚培养体系并对其理化特性进行了鉴定,从而为系统开展该病毒生物学特性和防控技术研究提供了必要的技术基础.

为建立一种灵敏、可快速检测猪圆环病毒4型(PCV4)的SYBR Green I实时荧光定量PCR方法,根据PCV4 Rep基因的保守区域设计引物,建立针对PCV4的SYBR Green I实时荧光定量PCR检测方法,并对该方法的特异性、灵敏性和重复性进行评价.结果 显示,该检测方法的Ct值与标准品在5.64×102～5.64×109 copies/μL范围内呈良好的线性关系,R2为0.999,斜率为-3.638,检测下限为5.64×102 copies/μL,且无非特异性扩增.利用该方法对56份临床样品进行检测,发现PCV4核酸阳性率为3.57％,比普通PCR更灵敏可靠.结果 表明,本试验建立的SYBR Green I实时荧光定量PCR方法可用于PCV4的快速诊断.

某竹鼠养殖场零星竹鼠出现不采食、拉稀等症状,并日渐消瘦至死亡.经实验室诊断为感染奇异变形杆菌,对患病竹鼠连续3d注射1 mg/kg头孢噻肟,病情有所控制.

禽致病性大肠杆菌(APEC)是一种能引起鸡、火鸡和其他鸟类肠外感染的致病性大肠杆菌,可以导致肉鸡气囊炎、败血型全身感染、蜂窝织炎和蛋鸡输卵管炎、腹膜炎.为了了解广西地区禽致病性大肠杆菌的耐药表型以及耐药基因的携带情况,本实验室对2019年从广西分离到的69株APEC采取K-B药敏纸片法进行药敏试验,药敏结果显示,69株APEC对氧氟沙星(56.5％)、恩诺沙星(69.6％)、氟苯尼考(79.7％)、氨苄西林(91.3％)、四环素(98.6％)耐药率较高,而对美罗培南、丁胺卡那霉素、呋喃妥因均不耐药;其中,多重耐药现象严重,对10种抗菌药物以耐4种、5种、6种的情况居多.同时用PCR扩增的方法对其耐药基因,包括碳青霉稀类、β-内酰胺类、氨基糖苷类、黏菌素类、喹诺酮类、四环素类在内的6大类共计17种耐药基因进行了检测.特别值得关注的是,发现了7株携带mcr-1基因的多黏菌素耐药APEC.药敏纸片法检测菌株的耐药表型和耐药基因存在一定关联度.本研究可为养禽场临床用药提供参考,同时为减缓耐药菌传播、降低对人类健康和公共卫生安全威胁提供依据.