Pseudorabies virus (PRV) is a highly pathogenic agent that adversely impacts swine populations, leading to considerable economic losses within the Chinese pig industry. Furthermore, the potential for PRV to transmit across species from pigs to other hosts has attracted significant attention. In light of this, it is essential to identify efficacious antiviral agents for the treatment of PRV infection. In this study, we investigated the antiviral properties of Diltiazem HCl (DTZ) against PRV infection in susceptible cell lines. Our results demonstrated that DTZ significantly inhibited PRV infection in both PK15 and Vero cells. Moreover, this chemical compound exhibited antiviral activity against both variant and classical strains of PRV, as well as herpes simplex virus type 1 (HSV-1). Time-of-addition assays showed that DTZ exerted its inhibitory effect through specific interference with the virus replication process. Subsequent transcriptomic analysis via RNA sequencing indicated that the calcium signaling pathway might be involved in the antiviral properties of DTZ against PRV infection. Specifically, treatment with EGTA or calcium ion (Ca2+)-free medium inhibited PRV infection; this inhibitory effect was substantially mitigated upon the reintroduction of CaCl2. In summary, DTZ effectively suppressed PRV infection in vitro, demonstrating its potential as an antiviral agent against PRV infections.

Toxoplasma gondii is a widely distributed intracellular parasite that disrupts host immune and metabolic homeostasis. Although accumulating evidence highlights the role of gut microbiota in parasitic infections, the effects of acute T. gondii infection on host gut microbial ecology remain poorly understood. In this study, metagenomic sequencing technology was used to systematically analyze the composition and functional alterations of the ileal microbiota in BALB/c mice on day 10 post-infection. Compared to uninfected controls, T. gondii infected mice exhibited a significant reduction in microbial diversity and a pronounced shift in community structure. Notably, there was an expansion of Proteobacteria, particularly the Enterobacteriaceae family, alongside a marked decline in beneficial taxa such as Actinobacteria and Bacillota. Functional annotation using the KEGG and CAZy databases revealed enrichment of metabolic pathways related to glycolysis/gluconeogenesis, O-antigen nucleotide sugar biosynthesis, bacterial secretion systems, and biofilm formation-Escherichia coli in the infected microbiota. These findings provide novel insights into the dysbiosis of gut microbiota and host-microbe interactions during acute T. gondii infection.

Maedi-visna (MV) and caprine arthritis encephalitis (CAE) are important viral diseases of sheep and goats. The diseases are caused by a group of genetically closely related lentiviruses known as small ruminant lentiviruses (SRLV), and are collectively referred to as SRLV infections. As the majority of sheep and goats infected with SRLV are asymptomatic, the disease is often overlooked. However, SRLV infection can significantly reduce animal productivity and impede animal trade. Currently, SRLV infection is widespread worldwide, but knowledge of its prevalence in China is limited due to a lack of cost-effective testing. In this study, we successfully developed an indirect enzyme-linked immunosorbent assay (iELISA) based on the SRLV capsid protein (CA) (p28) for the specific detection of anti-SRLV antibodies in serum. Using the application of checkerboard titration to optimize the iELISA assay conditions, the cut-off value was determined to be 0.09 by analyzing S/P values of 181 negative sera against SRLV that were confirmed with western blotting (WB). The method showed good reproducibility, with intra- and inter-assay coefficients of variation (CV) values less than 6.60%. A specificity test showed that the iELISA had no serological cross-reaction with six common small ruminant pathogens. Moreover, it was found to be 400-1600 times more sensitive than the available AGID test. The 93 clinical serum samples that tested SRLV-positive using the iELISA were all confirmed as positive using WB, indicating that the method has a low false positive rate. The iELISA was then applied to assess 4786 clinical serum samples from 13 cities in six provinces in China. The results show that the SRLV positivity rate of sera ranged between 0.85% and 40.00%, with the overall seroprevalence of SRLV being 10.64%. Our results indicate that the developed iELISA will serve as a valuable and efficient screening tool for the large-scale preliminary surveillance and monitoring of SRLV infections in both sheep and goats.

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

Antibody level detection is an important indicator for effectively evaluating animal epidemic prevention work,and strengthening the monitoring of antibody levels of certain animal diseases in the jurisdiction can provide a scientific basis for for-mulating animal disease immunization procedures and prevention and control measures.In order to understand the immune anti-body situation of foot-and-mouth disease in Zhaotong City from 2019 to 2022,the ELISA method was used to detect the lev-el of immune antibodies to foot-and-mouth disease O and A viruses in a total of 17210 sheep serum in nine counties and dis-tricts of Zhaotong City over a four-year period,and compared the immunization effects of foot-and-mouth disease in sheep in different counties and different years.In the nine counties and districts where samples were collected,the foot-and-mouth disease O-type antibodies were higher than the national level(＞70％);The antibody level of foot-and-mouth disease type A antibody in Sui-jiang County and Yiliang County did not reach the national level(＞70％).In Zhaotong City,the qualification rate of immune antibodies to FMD O in Zhaotong City has generally met the require-ments stipulated by the state,but the antibody level in some counties and districts is still at a rela-tively low level,and the immunization program should be adjusted on the original immunization program to achieve a better immunization level.The antibody levels of foot-and-mouth disease type A did not reach the national level in two coun-ties and districts,and the rest of the counties and districts met the national standard.Their immuni-zation schedules should be improved or more pro-tective vaccines replaced to increase levels of FMD type A antibodies in flocks and avoid economic los-ses due to foot-and-mouth disease type A virus infection.

2022年2月云南省元江县某农户饲养的山羊出现疑似羊痘病情,袭击率为16.91％(46/272),病死率为45.65％(21/46).为确定病因,防止病情扩散,开展了紧急流行病学调查.综合病羊临床症状以及现场调查和实验室检测结果,确诊本起疫情为输入性羊痘疫情.通过采取病羊与健康羊群隔开饲养、紧急免疫、对症治疗、消毒、加强饲养管理等干预措施,病情得到有效控制.分析认为,养殖户疫病防控意识淡薄,缺乏生物安全防范意识,调入携带病原羊只混群饲养等是引发病情的主要原因.本次病情警示,养殖场应加强生物安全管理,做好未出售羊群返场的隔离检疫,同时要加强饲养管理,减少应激因素,以预防疫病发生.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020–2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020–2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

为掌握目前市售小反刍兽疫-山羊痘二联活疫苗(Clone9株+AV41株)的免疫效果及抗体消长规律,分别选择云南玉溪市和楚雄市两地的健康羊群,皮内注射小反刍兽疫-山羊痘二联活疫苗,采集免疫后不同时间段的血清样品进行抗体检测,对疫苗的安全性和免疫效果进行评价.结果显示:除个别羊只体温略有升高、采食量略有下降外,其他羊只均未出现免疫应激反应和死亡的情况;用该疫苗免疫试验羊群,小反刍兽疫抗体阳性率高,建立了理想的免疫效果屏障,而羊痘抗体阳性率很低,不具有群体免疫保护作用或保护力低下.

为了解云南省马属动物弓形虫病流行情况,用夹心ELISA方法对云南省8个地区3种马属动物共969份血清样品进行弓形虫血清流行病学调查.结果弓形虫的抗体总阳性率为8.15％(79/969),其中马弓形虫抗体阳性率为3.03％(8/264),驴弓形虫抗体阳性率为8.92％(37/415),骡弓形虫抗体阳性率为11.72％(34/290).不同地区马属动物血清样本弓形虫阳性率差异不显著(P>0.05),不同种类马属动物血清样品弓形虫阳性率差异极显著(P<0.01).

为了解昆明滇池红嘴鸥(Larus ridibundus)携带的沙门氏菌(Salmonella spp.)的耐药性、毒力特征及致病性等情况,2019年11月,从昆明滇池随机选择100只越冬红嘴鸥,对其泄殖腔内容物进行细菌分离培养、生化鉴定、染色镜检、分子生物学鉴定、多位点序列分型和血清学鉴定;采用药敏纸片琼脂法对5大类14种抗菌药物进行耐药检测,使用PCR扩增检测耐药基因,分析耐药情况;通过毒力基因检测及动物致病性试验对所分离的细菌进行致病性分析.结果显示:确定其为ST19型鼠伤寒沙门氏菌(Salmonella Typhimurium),抗原结构式为O抗原1,4,[5],12;H抗原i,1,2,主要对阿莫西林、环丙沙星、四环素和多西环素耐药,且检出四环素类耐药基因tetB,喹诺酮类耐药基因gyrA、gyrB、parC和psrE.该分离菌检测出mogA、sseL、mgtC、bcfA、araB、spvC、spvB、stn、fimA、orgA、sopB、sipB和spaN 13种毒力基因.动物试验结果表明,该分离菌株对试验小鼠有一定致病性,接种浓度为5×106、5×107、5×108 cfu/mL的小鼠死亡率分别为60％、100％和100％,死亡小鼠出现肝脏肿大、肺部局部充血和肠壁变薄等病变.

为了解禽流感病毒(AIV)在野禽和家禽之间的传播特点,本研究于2020年3月～2021年3月在野生鸟类资源丰富的拉市海国际高原湿地采集到野鸟粪便样品1367份、家禽泄殖腔拭子420份和环境水样20份,共计1807份样品,通过鸡胚接种和RT-PCR分离鉴定出1株斑头雁来源的H9N2 AIV和2株家禽来源的H9N2 AIV.利用Illumina Miseq测序平台获得3株分离病毒的全基因组序列,并构建了其遗传进化树,分析了与流感病毒的传播能力及致病力相关的关键氨基酸位点的特征.遗传进化分析结果显示,野禽源和家禽源AIV具有相同的遗传进化特征:3株H9N2 AIV均属于欧亚谱系h9.4.2.5分支的病毒,其HA、NA基因为Y280-like亚群,PB2、M基因为G1-like亚群,PB1、PA、NP和NS基因为F/98-like亚群,推测3株分离株均为重组病毒,重组模式为中国流行的H9N2 AIV S基因型.氨基酸位点分析结果显示,3株分离病毒HA、NA、PB2、PB1、M1和NS1蛋白的突变位点均与病毒跨种属传播能力和小鼠致病力增强相关;同时野禽源H9N2 AIV同样存在家禽源分离株相似的HA蛋白N166D替换和NA蛋白颈部aa63～aa65位点缺失,但PA蛋白中未发生N383D突变.以上结果表明,本研究中野禽源H9N2 AIV可能源于家禽的传播,病毒在自然宿主野禽中发生了适应性突变.本研究首次对拉市海高原湿地野禽源与家禽源H9N2 AIV的遗传变异特征进行了系统分析,为禽流感的综合防控提供基础数据.

为了解云南省马属动物新孢子虫病流行情况,采用酶联免疫吸附试验检测了云南省8个地区的969份马属动物血清样品.结果显示,云南地区马属动物新孢子虫病抗体总阳性率为5.06％(49/969);其中马新孢子虫病抗体阳性率为1.08％(3/279),驴新孢子虫病抗体阳性率为7.25％(29/400),骡新孢子虫病抗体阳性率为5.86％(17/290);不同马属动物新孢子虫病抗体阳性率差异极显著(P<0.01);不同地区的马属动物新孢子虫病抗体阳性率差异显著(P<0.05);不同养殖模式的马属动物新孢子虫病抗体阳性率差异不显著(P>0.05).结果表明,云南部分地区马属动物中普遍存在新孢子虫感染,应采取有效的措施防控新孢子虫病.

Abstract Background Avian haemosporidia infect both domestic and wild birds, causing anemia, acute tissue degeneration, and depopulation in wild birds. Poultry and wild birds have been reported as common reservoirs of haemosporidia, but limited information is available for red junglefowl (Gallus gallus) in China. The present study investigated the prevalence and molecular characterization of haemosporidia in red junglefowl. Methods Blood samples were collected from 234 red junglefowl from Jinghong City of Yunnan Province, and genomic DNA was extracted from these samples. The prevalence of haemosporidia was determined by nested PCR targeting the mitochondrial cytochrome b (cytb) gene. Molecular characterization was investigated based on phylogenetic analysis of cytb sequences, and associated risk factors were analyzed using the Chi-square (χ 2) test. Results The overall prevalence of haemosporidia was 74.8% (175/234), and three species were identified, namely Haemoproteus enucleator, Leucocytozoon californicus, and Plasmodium juxtanucleare. The prevalence of haemosporidia in adult fowl (81.1%, 107/132) was significantly higher (χ 2 = 6.32, df = 1, P = 0.012) than that in juveniles (66.7%, 68/102). Three novel haemosporidian lineages were revealed. Conclusions This study examined the prevalence and identified species of avian haemosporidians in red junglefowl, providing new information on the molecular epidemiology and geographical distribution of haemosporidian parasites. Our results indicated high prevalence and diverse species distribution of these haemosporidians in red junglefowl. To the best of our knowledge, this is the first record of haemosporidian infection in red junglefowl in China. Graphical Abstract

为鉴定山羊脑多头蚴,通过对虫体进行形态学观察并采用PCR扩增虫体线粒体细胞色素C氧化酶亚基1(cox1)基因,将有效序列进行NCBI-Blast在线比对,利用MEGA6.0构建遗传系统进化树.结果 显示,山羊脑多头蚴cox1序列长度为446 bp,与多头带绦虫同源性达100％,系统进化分析显示分离株与多头带绦虫位于同一分支.结合形态学观察结果表明,山羊所感染的多头蚴属于多头带绦虫幼虫——脑多头蚴,cox1基因可作为鉴定多头带绦虫的有效分子标记,研究结果为山羊脑多头蚴的分子诊断提供参考依据.

以某濒危动植物收容拯救中心死亡狐狸(Vulpes sp.)病例为研究对象,对其进行临床检查,尸体病理剖检,肠道组织及内容物实验室病原学检测.结果显示:临床症状可见该狐狸体表皮肤正常,身体消瘦,毛发杂乱无光泽,肛门直肠脱出,尾根部有血迹;尸体剖检观察可见该狐狸胃中无内容物,肝脏有大面积的点状出血、脾脏有明显的大面积出血面并伴有梗死灶,结肠前段有一处肠扭转、结肠中后段有2处约为8 cm的套叠,套叠肠管淤血、坏死,肠内有出血,套叠肠段之间的肠管有水肿,其余组织器官无明显病理变化;实验室检测犬瘟病毒(canine distemper virus,CDV)、冠状病毒(canine Coro-naviridae,CCV)、轮状病毒(canine rotavirus,CRV)呈阴性,细小病毒(canine parvovir-us,CPV)检测结果呈阳性.综合分析结果表明,该狐狸因感染细小病毒致死与肠套叠密切相关,后续该类疾病的治疗要引以为鉴.

为了解H5N2亚型禽流感病毒(Avian influenza virus,AIV)的血凝素蛋白(Hemag-glutinin,HA)系统进化特征,借助NCBI influenza virus resource数据库,下载禽源A型流感病毒(Influenza A Virus,IAV)H5N2亚型HA核酸序列,分析毒株的宿主、分离时间等特征,并构建HA蛋白的遗传进化树,分析其系统发生.结果显示:NCBI数据库收录的H5N2 AIV的HA核酸序列1 272条,序列在NCBI发布时间集中于2005-2007年、2011年、2014-2016年;582条序列为野禽来源,宿主主要分布于雁形目、鸡形目和鹆形目的野禽.系统发生分析结果表明:H5N2 AIV的HA蛋白可聚类为亚欧谱系及北美谱系两大分支,前者可分为三个亚系,后者可分为两个亚系.研究结果呈现了H5N2 AIV HA基因在时间及空间上的进化特征,为H5N2 AIV引起的疫病预警和防控提供理论基础.

2020年1月3日,某猪场部分生猪出现腹泻症状,3 d后达发病高峰,波及全场,并伴有部分生猪死亡,袭击率为60.66％,病死率为6.88％.为探寻病因,采取现场调查与实验室检测相结合的方式,对此次猪群腹泻进行了调查,综合推断此次暴发是由大肠杆菌、沙门氏菌、志贺氏菌引起的细菌性腹泻及由应激因素引起的应激性腹泻,饮入污水、断水应激、寒冷应激是导致本次猪群腹泻的主要原因.据此,提出了拌料饲喂抗生素、清洗消毒圈舍和饮水设施、强化饲养管理等干预措施,并有效控制了疫情.调查提示,加强饲养管理,减少寒冷、缺水引起的应激,保持清洁饮水,对预防猪群腹泻具有十分重要的意义.

布鲁氏菌病(brucellosis,简称布病)呈世界性流行,全球有超过170个国家有布病报告.20世纪90年代中后期布病呈回升趋势,进入21世纪后疫情更加严峻;感染途径由主要的职业接触感染扩展到非职业的食源性感染,发病区域由北方牧区逐步向周边的半农半牧区和农区扩散,并向南方传播扩散.大多数患者通过直接或间接接触受感染的动物或动物产品感染,大多数动物感染是从患病动物、受污染的饲料和饮水点摄入病原体所致.目前,布病检测方法较多且各有其特点,其中细菌分离培养是"金标准",补体结合试验(CFT)是OIE公认的确诊试验.疫苗免疫是布病最为有效的控制措施,但目前尚未有国际认可的用于人的布病疫苗,动物疫苗也存在安全性差、免疫期短等多种缺陷.未来需要加强布病监测并制定有效的防治措施,开展多学科、多部门、多领域的携手合作,共同推动布病的预警、监督和防控.

为了解野生水禽绿鹭(Butorides striata)中分离到的1株H9N2亚型禽流感病毒(A/striated heron/Yunnan/2018)的生物学特性;对其进行全基因组序列扩增、测序、进化分析;序列分析显示:该分离株HA、NA基因位于Y280-like分支、PB2、M基因位于G1-like分支、PA、PB1、NP、NS基因位于F98-like分支,分别与H9、H7、H10等多种亚型的AIV同源性较高,该分离株不同基因片段来源较复杂.HA裂解位点氨基酸序列为333PSRSSR↓GL340,符合低致病性禽流感病毒(LPAIV)氨基酸序列特征;S145N突变增加了一个糖基化位点,提示该位点出现可能会使毒株致病性提高,免疫原性发生改变;HA受体结合位点发生Q234 L突变,表现出人流感病毒受体结合特性;NA基因出现第63—65位氨基酸缺失,M1发生N30D,T215A突变,M2发生S31N的突变,PB2、PB1、NS、NP、PA关键位点未发生变化,分析结果提示当前分离株已出现耐药性、致病性增强的变化.本研究表明该分离株呈现遗传演化的多样性及基因重组的复杂性,因此加强对野生水禽类禽流感病毒的监测和研究具有重要的公共卫生意义.

试验旨在研究核桃青皮水取物对云南本地土杂鸡生长性能和禽流感、新城疫抗体水平的影响.选取120只28日龄本地土杂鸡,随机分为4个组(对照组、试验1组、试验2组、试验3组,分别在饮水中添加0、1％ 、3％ 、5％ 核桃青皮水提物),每组30只(公母各半),1只为1个重复.生产性能测定结果表明:65日龄时,各试验组平均体重均显著高于对照组(P<0.05),且试验3组的明显高于其他试验组(P<0.05);试验3组平均日增重(ADG)为62.89 g,明显高于其他各组(P<0.05),料重比(F/G)也明显低于对照组和其他试验组,分别降低了12.00％ 、13.39％ 、5.17％(P<0.05).新城疫(ND)和禽流感(H7、H5)抗体水平的测定结果表明,免疫46 d后试验3组鸡新城疫抗体水平明显高于对照组和其他试验各组(P<0.05).免疫48 d后试验2和3组鸡禽流感(H7)抗体水平显著高于对照组和试验1组(P<0.05).试验2和3组禽流感(H5)抗体水平与对照组无明显差异(P>0.05).试验结果表明,核桃青皮水提物可提高云南本地土杂鸡的生长性能和新城疫、禽流感(H7)抗体水平,且其效果随着核桃青皮水提物饮水浓度的增加而提高,综合来看以5％ 核桃青皮水提物效果最好.