IntroductionViral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea.MethodsSpecific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods.ResultsThis novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R2 > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region.DiscussionThe mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

Listeria monocytogenes (LM) is a zoonotic pathogen that causes sporadic infectious listeriosis, which is a foodborne disease associated with consumption of contaminated food or feed. The internal organs of an African lion from a zoo in Shanghai were analysed to determine the cause of death. LM infection was suspected on the basis of the clinical symptoms and pathological changes and confirmed by polymerase chain reaction, whole genome sequencing and phylogenetic analysis. This is the first report of LM infection of an African lion in China.

Urban stray cats are cats without owners that survive in the wild for extended periods of time. They are one of the most common stray animals in cities, and as such, monitoring the pathogens carried by urban stray cats is an important component of urban epidemiological surveillance. In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we collected 374 ocular, nasal, and oropharyngeal swabs from urban stray cats in Shanghai from January 2022 to December 2022. After RNA extraction, we used real-time PCR to detect six respiratory pathogens, including influenza A virus, feline calicivirus, feline herpesvirus type 1, Mycoplasma, Chlamydia, and Bordetella bronchiseptica. The results showed that among the 374 samples, 146 tested positive, with a positivity rate of 39.04%. The highest positivity rate was observed for Mycoplasma felis at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus 1 at 3.48% (13/374), Bordetella bronchiseptica at 1.34% (5/374), and influenza A virus was not detected. The highest positivity rate for Mycoplasma felis was in Minhang District at 31.94% (23/72), while Chlamydia felis and Bordetella bronchiseptica had the highest positivity rates in Jiading District at 23.53% (8/34) and 5.88% (2/34), respectively. The highest positivity rates for feline calicivirus and feline herpesvirus 1 were both observed in Qingpu District, at 14.46% (12/83) and 9.64% (8/83), respectively. A total of 36 samples showed mixed infections with two or more pathogens, with Mycoplasma felis being involved in 32 of these mixed infections, with the highest number of mixed infections being with Chlamydia felis at 25 samples. Respiratory pathogen positivity was detected throughout the year, with peak detection rates in summer and winter. The positivity rates of cat respiratory pathogens in different seasons showed statistical differences (χ2 = 27.73, p < 0.01). There was no statistical difference in the positivity rates of respiratory pathogens between cats of different genders (χ2 = 0.92, p > 0.05). The positivity rates of respiratory pathogens in cats of different age groups showed statistical differences (χ2 = 44.41, p < 0.01). Mycoplasma felis and Chlamydia felis were the main pathogens causing respiratory infections in stray cats, with Mycoplasma felis showing a much higher positivity rate than other respiratory pathogens and often co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was high in summer, autumn, and winter, with no statistical difference between seasons. These results indicate a serious overall prevalence of respiratory pathogens in urban stray cats in the Shanghai area, showing seasonal trends and mixed infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area.

Our study was designed to investigate the original spectrum of feline respiratory tract infection and to provide a scientific basis for the clinical diagnosis and treatment of feline respiratory infections and for precise prevention and control measures. A total of 400 cats with upper respiratory tract infections from animal hospitals in 12 provinces in China were examined from November 2022 to October 2023 to investigate the epidemiology of feline calicivirus (FCV), feline herpes virus type 1 (FHV-1), influenza A virus (IAV), Mycoplasma felis, Chlamydia felis, and Bordetella bronchiseptica through loop-mediated isothermal amplification (LAMP) with microfluidic chip detection. The results showed that 396 of the 400 samples tested were positive for at least one of these pathogens, with an overall detection rate of 99.00

随着"互联网+"技术快速发展,实验室信息管理系统(laboratory information management system,LIMS)在兽医检测行业中应运而生.L IM S的应用使得兽医实验室检测业务的管理变得标准化、信息化、科学化,从而促进了检测业务的高效运行.本文阐述了L IM S在兽医实验室检测业务管理中应用的现状、优势及存在问题,并提出了几点建议,以期促进L IM S更好地服务于兽医检测行业.

IntroductionPullorum disease is one of the common bacterial infectious diseases caused by Salmonella pullorum (S. pullorum), which can result in a decrease in the reproductive performance of laying hens, thus causing considerable economic losses. However, studies about the characteristics of intestinal microbiota with pullorum and their potential association with reproductive performance in hens are still limited. This study was to identify the gut microbiota associated with S. pullorum in poultry.MethodsA total of 30 hens with S. pullorum-negative (PN) and 30 hens with S. pullorum-positive (PP) were analyzed for hatching eggs laid in 2 weeks (HEL), fertilization eggs (FE), chick number (CN), and microbial structure.ResultsThere were significant differences in HEL (p < 0.01), FE (p < 0.01), and CN (p < 0.01) between PP and PN. Histomorphological observations showed abnormal morphology of the ovaries and fallopian tubes and low integrity of epithelial tissue in the ileum and cecum in PP. 16S rRNA gene sequencing revealed that beneficial cecal microbes, such as Bacteroides, Desulfovibrio, and Megamonas, were positively correlated with reproductive performance and had lower abundance in PP (p = 0.001). Furthermore, diminished phosphotransferase system (PTS) and pentose phosphate pathway, butanoate metabolism and oxidative phosphorylation were also found in PP.DiscussionTaken together, this study clarified the morphological characteristics of the reproductive tract and intestines of chickens infected with S. pullorum and preliminarily explored the potential association between cecal microbiota and reproductive performance in hens. Our data may provide a reference for revealing the intestinal microbial characteristics of hens in resisting pullorum and exploring novel approaches to infection control in future studies.

农业现代化是实现乡村振兴战略的重要举措,着力提升现代畜牧业发展水平对加快建设农业强国、推进农业现代化建设具有重要意义.上海市浦东新区以发展大都市小农业中的精品畜牧业为基本定位,在畜禽养殖生态化、疫病区域化管理、畜禽养殖标准化率方面取得新成效;但也面临基层政府支持不够、产业结构发展不均衡、畜牧业发展土地利用空间受限、对企业经营的保护性措施不足、涉农人才支撑力度不够等问题.因此,需要增强属地政府发展原动力、落实产业发展建设用地、稳定健全产业扶持政策及人才兴农强农政策、加大金融支持力度、提升科技创新驱动能力、立足种源做长产业链,以促进浦东新区都市型现代畜牧业的健康发展.

为诊断一起疑似湖羊细菌感染导致的急性死亡病例,在流行病学调查、临床诊断基础上,开展实验室细菌病原学检测.从病死羊内脏组织中分离到3种不同的细菌,发现菌株的生化反应均较弱,无法通过传统的生化鉴定得出准确的结果,经MALDI-TOF MS鉴定为多杀性巴氏杆菌、溶血曼氏杆菌和绵羊链球菌,并用PCR方法进行了验证.综合临床诊断、细菌分离与药敏试验分析,最终认定绵羊链球菌为此次湖羊发病死亡的主要病原,经针对性治疗,疫情在2 d后得到有效控制.说明绵羊链球菌可对湖羊致病,同时还具有较强的耐药性,是羊群的一种潜在的致病菌,应给予足够的重视.

猫皮肤肥大细胞瘤是猫第二常见的皮肤肿瘤,常发于中老年猫,病例数约占猫科动物皮肤瘤的 20%,目前在我国文献中鲜有报道.本文介绍了近期临床接诊的一例猫肥大细胞瘤病例.患猫体表肿物位于右耳根部,呈粟粒样突起,无游离性;血常规及血液生化检查结果显示,患猫单核细胞数目、百分比下降,中性粒细胞百分比上升,球蛋白含量上升;H.E.染色可见病变位于表皮下真皮层内,病变细胞为卵圆形,呈弥漫性浸润性生长,排列成条索状或巢团状,细胞核呈圆形,胞质较丰富.综合临床检查、血常规检查、血液生化检查以及病理组织细胞学检查结果,体表肿物被诊断为猫肥大细胞瘤.经手术切除后,对患猫进行消炎抗菌、抗组胺以及肿瘤靶向药物治疗,1月后复检,患猫痊愈.该病例可为猫肥大细胞瘤的临床诊断与治疗提供参考.

牛源致病性大肠杆菌是导致牛细菌性疾病的重要病原体之一,抗菌药是治疗及预防牛大肠杆菌病的重要手段,然而在养殖过程中,长期且不合理的使用抗菌药物,导致大肠杆菌耐药问题频发,且耐药性具有通过人与动物的直接接触或借助食物链将耐药基因从动物传播给人类的可能,严重威胁人类健康及公共卫生安全.本文根据近几年国内外关于牛源大肠杆菌耐药性基因的研究报道,对大肠杆菌耐药机制、耐药基因检出率及耐药表型进行综述,以期为大肠杆菌耐药性的研究提供参考.

HPS_06257 has been identified as an important protective antigen against Glaesserella parasuis infection. However, little is known about the role of HPS_06257 in the protective immune response. A whole-genome data analysis showed that among 18 isolates of Glaesserella parasuis, 11 were positive for the HPS_06257 gene, suggesting that not every strain contains this gene. We used PCR to investigate the presence of the HPS_06257 gene among 13 reference strains and demonstrated that 5 strains contained the gene. A polyclonal antibody against HPS_06257 was generated with a recombinant protein to study the expression of HPS_06257 in those 13 strains. Consistent with the PCR data, five strains expressed HPS_06257, whereas eight strains were HPS_06257 null. We also compared the protective effects of HPS_06257 against an HPS_06257-expressing strain (HPS5) and an HPS_06257-null strain (HPS11). Immunization with HPS_06257 only protected against HPS5 and not HPS11. Moreover, phagocytosis of antibody-opsonized bacteria demonstrates that the antibody against HPS_06257 increased the phagocytosis of the HPS5 strain by macrophages but not the phagocytosis of the HPS11 strain, suggesting that antibody-dependent phagocytosis is responsible for the protective role exerted by HPS_06257 in the immune response to HPS5. Our data also show that the antibody against HPS_06257 increased the phagocytosis of the other HPS_06257-expressing strains by macrophages but not that of HPS_06257-null strains. In summary, our findings demonstrate that antibody-dependent phagocytosis contributes to the protective immune response induced by immunization with HPS_06257 against HPS_06257-expressing strains.

为了解上海市猪丹毒杆菌流行株的耐药状况,对上海市2011—2017年分离到的猪丹毒杆菌采用纸片扩散法(K-B法)和微量肉汤稀释法(MIC法)进行药敏试验,同时对临床分离株与健康带毒株的MIC结果进行比较.结果显示:所有菌株对β-内酰胺类和大环内酯类药物敏感,而对林可霉素、四环素类、氯霉素类、喹诺酮类等原本敏感的药物,已产生较强耐药性;对临床分离株与健康带毒株的MIC结果比较分析发现,致病菌株和健康带毒株的耐药性存在较大差异.结果表明,上海市猪丹毒杆菌耐药情况较为严峻,除对β-内酰胺类和大环内酯类药物较为敏感外,对其他药物均出现不同程度的耐药.本研究为临床合理用药提供了有价值的参考资料.

为了解上海市生乳中肺炎克雷伯氏菌污染水平,分析本市乳制品中肺炎克雷伯氏菌污染风险来源及耐药情况,本研究对上海市乳品加工厂的生乳样本和养殖场环境样本进行了肺炎克雷伯氏菌计数和细菌分离鉴定,并对分离菌株开展毒力基因检测和耐药性分析.结果显示,上海市健康奶牛中肺炎克雷伯氏菌分离率为11.25％,远低于患乳房炎奶牛样品检出率;环境溯源样品中肺炎克雷伯氏菌检出率为21.3％,多在垫料及土壤中检出;对80家奶牛场未患乳房炎奶牛样品和47批次环境溯源样品细菌分离结果均表明,夏季肺炎克雷伯氏菌检出率显著高于冬季;本市分离的肺炎克雷伯氏菌,对β-内酰胺类和氨基糖苷类药物存在普遍耐药,且多重耐药性十分严重,耐药菌株占比达98.5％;分离菌株中毒力基因mrkD、fimH、wabG的检出率分别为88.89％、66.67％、33.33％,rmpA未检出.以上结果表明,夏季为肺炎克雷伯氏菌高发季节,且主要的外部污染风险为垫料和土壤,毒力基因检测表明,菌株不具备高致病性,多为呼吸道和消化道常在菌.通过本研究,提示居民不宜直接饮用生乳,养殖场应加强养殖环境、挤奶及生产运输环节的卫生管理,不滥用抗生素.

2020年11月,上海市某宠物医院接诊一例疑似猫慢性牙龈炎(feline chronic gingivostomatitis,FCGS)病例.为确定病因,采集该病例的眼、口、鼻拭子进行病原分离鉴定,结果确定为猫杯状病毒(feline calicivirus,FCV)和细菌的混合感染.对FCV分离株VP1基因进行扩增和测序分析,发现其与其他18株序列的核苷酸同源性为74.2％～81.0％,编码蛋白氨基酸同源性为82.2％～88.1％.构建进化树分析发现,FCV分离株与国内毒株的遗传关系较国外更近,但与目前临床上使用的疫苗株处于不同的进化分支.对VP1蛋白D区和E区分析发现,D区及conE区的线性表位较为保守,而E区的5'HRV表位存在较大变异,与抗体反应有关的第439～441位氨基酸同样变异较大.本研究为了解FCV的变异特点以及该病的临床防治提供了参考.

为了解上海市生乳中金黄色葡萄球菌污染水平,分析上海市乳制品中金黄色葡萄球菌污染风险来源及乳制品安全情况,本研究对该市乳品加工厂的生乳样本进行了金黄色葡萄球菌计数和细菌分离鉴定,并对分离菌株开展了毒力基因和耐药基因检测.结果显示,84份生乳样品的计数检出率为14.29％,计数范围为20～190CFU/mL,中位数为50CFU/mL.上海市生乳样本金黄色葡萄球菌核酸阳性率38.1％,菌株分离率27.38％.养殖投入品样本中未检出金黄色葡萄球菌.144份养殖环境样本中,仅乳头奶、乳房表面拭子和垫料中检出.分离菌株中肠毒素检出率仅为8.70％,耐甲氧西林金黄色葡萄球菌(MRSA)检测均为阴性.表明上海市生乳中存在一定的金黄色葡萄球菌污染,不宜直接饮用,但该污染水平较低;饲养环节中投入品污染风险较小,饲养环境为乳品中金黄色葡萄球菌污染的主要来源.提示养殖环境控制是防止生乳金黄色葡萄球菌污染、防控奶牛乳房炎的一项重要手段.

为了解上海地区种猪场猪群猪链球菌流行情况、血清型分布情况,于2020年5月,从上海8家规模化种猪场采集猪口液样品148份,开展猪链球菌监测及血清型分型研究.86份检测口液样品猪链球菌阳性,阳性率58.11％.86株猪链球菌分离株中,49株鉴定为传统血清型,血清型鉴定率56.98％;共鉴定出12种血清型,其中优势血清型为猪链球菌7型(42.86％),其次为12型(20.41％),1型和2型猪链球菌各检出1株,9型未检出.本次调查结果提示,上海地区健康猪群携带的1型、2型、7型猪链球菌可能成为感染人和猪群的风险因素.

To construct a protein fingerprint database of Haemophilus parasuis (H. parasuis), thus improving its clinical diagnosis efficiency. A total of 15 H. parasuis standard strains were collected to establish a protein fingerprint database of H. parasuis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the effects of different culture media and culture time on the quality and identification results of the protein fingerprint were investigated. The results showed that tryptone soy agar (TSA) and tryptone soy broth (TSB) media and different incubation times had no significant effect on the characteristic peaks of the protein profiles. In addition, 18 clinical isolates were used to compare the identification results of the self-built protein fingerprint database, PCR detection, and basic database. Only one strain was identified in the original VITEK-MS system database, while the self-made protein fingerprint database of H. parasuis was 100% accurate for the detection of 18 clinical isolate strains. The protein fingerprint database of H. parasuis built by our laboratory is suitable for rapid clinical diagnosis of H. parasuis, due to its high accuracy, efficiency, and strong specificity.

为确定上海地区某雉鸡场雉鸡发病死亡的原因,对病鸡进行剖检和细菌分离,并应用基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)蛋白质谱和分子生物学方法(测定分析16S rDNA序列并与参考序列进行同源性比对分析)对菌株进行鉴定.结果:分离菌株ph20为多杀性巴氏杆菌杀禽亚种荚膜A型.小鼠攻毒实验证实,该菌株毒力较强.结合病死雉鸡的临床症状和病理变化,确诊病例为雉鸡禽霍乱.

从上海的1只无症状流浪猫的全血中分离到1株细菌,综合培养性状、生化特性,该菌株鉴定为大肠埃希菌(Escherichia coli),经PCR检测和O抗原血清型试验鉴定为产肠毒性大肠埃希菌O78:k80.该菌株对林可霉素耐药,对阿莫西林、氨苄西林、卡那霉素等15种抗生素均敏感.利用该菌株接种ICR小鼠,试验小鼠20 h内死亡.

利用PCR结合变性高效液相色谱法快速检测大肠埃希菌喹诺酮耐药决定区gyrA基因和gyrB基因突变.方法:对22株喹诺酮耐药大肠埃希菌进行2种基因的PCR扩增,产物经DNA杂交形成同源和异源双链后进行变性高效液相色谱法检测和序列测定.结果:20株试验菌gyrA基因出现异常峰型,14株试验菌gyrB基因出现异常峰型.测序证实所有异常峰型的样本均存在突变位点,正常单峰则无突变位点.其中gyrA基因存在Ser83Leu和Asp87Asn突变,突变率分别为90.9％和86.4％;gyrB基因存在Glu185Asp突变,突变率为4.5％,两种基因均存在多个位点的同义突变.结论:DHPLC方法可用于快速检测大肠埃希菌gyrA和gyrB基因突变.