In this study, we used baculovirus to express hemagglutinin (HA) and neuraminidase (NA) to prepare a novel genotype of H3N2 canine influenza virus particles (VLPs). The effectiveness of the H3N2 VLP vaccine was evaluated by detecting HI antibodies, the antiviral protection rate, antibody persistence and anatomical examination of the lungs.A challenge model has been established in a previous study for the study of canine influenza virus-like particle vaccines. A/Canine/Shanghai/0103/2019, with a challenge dose of 106 EID50, infects 10week-old healthy beagle dogs through nasal instillation and can cause severe clinical symptoms. Using a single dose of VLP vaccine for beagle dogs, the vaccine was tested at titers of 26 intranasally and 26 intramuscularly. One week after a single immunization, the HI titer promptly reached 28 among the immunized groups. The duration of antibody can persist for four months. We differentiated between CD4+ and CD8+ T cells in the peripheral blood. Four weeks after the single immunization, all beagles except those in the noninfected and nonimmunized groups were intranasally challenged with live H3N2 virus (1 x 106 EID50). All immunized beagles shed no virus at d 1-4 post-challenge. After the challenge, the placebo control beagles shed the virus on d 1 postchallenge (105.85 +/- 0.071 EID50). An anatomical examination of the lungs revealed that visible lesions were rarely detected in the lungs of the nasal immunization group, and the lungs were as healthy as those of the noninfected and nonimmunized groups were. The lung surfaces presented visible bleeding spots in the intramuscular immunization group and placebo-control group. Their effectiveness will provide a scientific basis for the promotion and use of these products.

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.

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.

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.

In this study, one G2c-subtype strain of porcine epidemic diarrhea virus (PEDV) (SHXX1902 strain) was isolated from clinical samples in suspended Vero cells, which was different from the genotype of the commercial AJ1102 vaccine. As a result, we determined the pathogenicity of different passages' isolates (SHXX1902 strain) and compared the immunogenicity of G2c-subtype strain (SHXX1902 strain) with the commercial AJ1102 vaccine. The viral titer reached 10(7) 50% tissue culture infectious dose (TCID50)/ml, which met the requirement for seed virus replication during vaccine development. Five-day-old piglets were orally infected with viruses from passages P5 and P35 to determine the pathogenicity and immunogenicity of different passages. Pregnant sows were immunized with inactivated SHXX1902-P5 or the commercial AJ1102 vaccine (first immunized with an attenuated vaccine and then boosted with an inactivated vaccine) to study the influence of the culture method on the immunogenicity of the strain. The median pig diarrhea dose (PDD50) and the median lethal dose (LD50) of the P5 virus were 10(2.00) and 10(2.84) TCID50/ml, respectively. All five piglets infected with the SHXX1902-P5 virus shed the virus 24 h after vaccination, whereas only two of the five piglets treated with the SHXX1902-P35 virus shed the virus 48 h after vaccination. The SHXX1902-P35 virus was partially attenuated in the 5-day-old piglets. Inactivated SHXX1902-P5 induced PEDV-specific immunoglobulin G (IgG) antibody responses equivalent to those induced by AJ1102 after infection in sow serum. However, the IgA titer induced by AJ1102 was much higher than that induced by inactivated SHXX1902-P5 since the boost immunization. On days 5 and 7 after farrowing, the IgA titers were similar among the immunized groups. Our study highlights that serial passage can lead to the attenuation of G2c-subtype strain. The immunogenicity of the inactivated strain was similar to the commercial vaccine. Our observation helped conceptualize appropriate study designs for the PEDV vaccine.

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.

为了解上海地区动物源性金黄色葡萄球菌的耐药情况,采用微量肉汤稀释法对1999年、2011—2018年(2016年除外)分离鉴定保存的78株金黄色葡萄球菌进行19种药物的药敏试验,采用头孢西丁纸片法进行耐甲氧西林金黄色葡萄球菌(Methicillin-resistant staphylococcus au-reus,MRSA)菌株筛选,采用聚合酶链反应(PCR)进行mecA基因检测.结果:金黄色葡萄球菌除万古霉素、利奈唑胺、夫西地酸、莫匹罗星外,均产生耐药性,多重耐药现象严重,耐药谱广;MRSA检出率为51.3％,发病样品、健康畜禽的粪便样品、环境样品中均检测到MRSA;mecA基因检出率为51.3％,MRSA中mecA基因携带率显著高于甲氧西林敏感金黄色葡萄球菌(Methicillin-sensitive staphylococcus aureus,MSSA).

为调查上海市猪链球菌耐药情况,采用体外最小抑菌浓度(MIC)药物敏感试验,对2004—2017年从上海市分离的150株猪链球菌,开展了5类8种临床常见抗菌药物的敏感性检测.结果显示:95％ 的猪链球菌对8种抗菌药物产生了不同程度的耐药,耐药率为16.67％～90.00％.对 β-内酰胺类药物的耐药性较低,耐药率分别为氨苄西林16.67％、青霉素19.33％、头孢噻呋26.00％,但也表现出耐药性加重的趋势;对酰胺醇类和喹诺酮类药物出现了一定程度的耐药,其中对氟苯尼考的耐药率为24.67％,对环丙沙星和氧氟沙星的耐药率均为42.00％;对大环内酯类和四环素类药物则出现了极高的耐药性,其中对红霉素、四环素的耐药率分别达到82.67％、90.00％.46％ 的菌株为多重耐药,其中10.7％ 的菌株为8重耐药.结果表明,上海市猪链球菌耐药情况较为严峻,除对 β-内酰胺类药物较为敏感外,对其他药物均出现了不同程度的耐药,且普遍为多重耐药.因此,上海市需加强兽用抗菌药物的临床用药指导,倡导合理规范用药.

某猪场断奶仔猪转保育舍后,出现精神委靡、消瘦直至死亡临床症状.对该猪群病死仔猪进行病理剖检,无菌采集心、肝、脾、肺、胆汁进行病原学检测,通过细菌分离鉴定及VITEK等方法鉴定出该仔猪感染支气管败血波氏杆菌,并通过15种药物的药敏试验,发现该细菌对阿莫西林、环丙沙星等敏感,通过荧光PCR方法检测到猪繁殖与呼吸综合征核酸阳性.根据诊断结果,建议用弱毒苗或基因缺失疫苗进行紧急免疫,结合补充电解多维、淘汰隔离和消毒等综合措施对该猪群进行治疗.

为了解上海市活禽交易市场鸡群中沙门氏菌的携带情况,我们于2016年对市区的8个活禽交易场所进行了调查.共采集肛门拭子500份,分离培养沙门氏菌,运用诊断血清、VITEK 2 Compact和VITEK MS分别对分离菌株进行血清型、生化和质谱鉴定,使用PCR和药敏板分别对分离菌株的耐药基因和耐药性进行检测.结果显示:从500份肛门拭子中分离鉴定出28株沙门氏菌,分离率为5.6%,以肠炎沙门氏菌、印第安纳沙门氏菌和鼠伤寒沙门氏菌为主,说明在市场交易环节应当加强对沙门氏菌的防控;耐药基因检出率较低,仅有喹诺酮类和磺胺类抗生素耐药基因有检出,分别为21.4%和7.1%,I类整合酶基因检出率达到28.6%;从耐药性检测结果看,分离菌株对磺胺类抗生素、头孢噻肟和庆大霉素的耐药率达到50%以上,对环丙沙星、美罗培南、萘啶酸、头孢他啶和氨苄西林的耐药率在40%以下,这些药可用于鸡群沙门氏菌的防控;耐药基因和耐药性之间无明显相关性.

[目的]了解1997—2010年上海市猪源大肠杆菌的耐药情况和产ESBLs大肠杆菌的基因型分布.[方法]采用K-B法对199株猪源大肠杆菌进行药敏试验,采用PCR方法对TEM、CTX-M、SHV和OXA等4种基因型进行检测.[结果]199株猪源大肠杆菌对20种抗菌药物均表现出不同程度的耐药性.耐药率最低的为大观霉素(7.04％),其次为头孢曲松(10.55％)、头孢哌酮(12.56％),最高的为林可霉素(100％),对阿莫西林和复方新诺明的耐药率分别高于80％和93.33％.5～15耐的菌株占88.94％.15种抗菌药物之间均存在不同程度交叉耐药性.共检出15株产ESBLs菌株(7.54％),其中2株TEM型,5株CTX-M型,7株同时携带TEM、CTX-M型.[结论]上海市猪源大肠杆菌分离株耐药谱广、耐药率高;对抗菌药物的交叉耐药现象严重,交叉耐药率有的相似,有的差异大.猪源大肠杆菌ESBLs基因型主要以TEM型和CTX-M型为主,尚无SHV型和OXA型.

目的:建立猪链球菌的快速计数方法.方法:采用分光光度法进行细菌计数方法研究,以平板计数法测定菌液中细菌总量,并结合OD600研究两者之间的线性关系.结果:分别对猪链球菌5个不同的稀释度进行OD600检测,同时对菌液进行平板计数获得对应细菌总数,以600 nm处吸光度(OD600)作为纵坐标,以平板计数的细菌数(cfu)作为横坐标建立猪链球菌细菌数与吸光度相关关系的标准曲线.结论:经验证,建立的猪链球菌分光光度测定法可用于菌液浓度的快速测定,便于工作快速有效开展.

2016年2月,上海市崇明区某乡镇一规模养猪场出现40多头5~8周龄仔猪发病死亡情况.经细菌分离、分离菌株生化鉴定和PCR检测,确诊为猪链球菌2型感染.对该菌株采用K-B纸片法,进行10种常用抗菌药物的体外药敏试验,结果发现该细菌对青霉素、阿莫西林、恩诺沙星敏感,对诺氟沙星、阿奇霉素、壮观霉素、卡那霉素、阿米卡星、庆大霉素和氨苄西林不敏感.根据药敏试验结果,建议养猪户使用阿莫西林和恩诺沙星进行治疗,并做好病猪的隔离与护理.一周后病情得到控制.

为了解上海及周边地区猪源沙门氏菌的血清型分布、毒力基因及分子分型情况，我们对21株临床分离到的猪源沙门氏菌用沙门氏菌属诊断血清鉴定血清型；采用多重聚合酶链式反应，检测17个主要的毒力基因；应用脉冲场凝胶电泳（PFGE）技术进行分子分型，探寻21株沙门氏菌之间的亲缘关系。结果显示，21株沙门氏菌中，19株血清型为7∶c∶5，1株为4∶i∶2，1株未鉴定出；21株沙门氏菌的17个毒力基因中检出16个，菌株之间无差别；经PFGE后，每个菌株产生13～18条电泳条带，用Bionumerics软件进行聚类分析表明，21株猪源沙门菌相似度在77％～100％，可分为2种不同的基因型，其中A型有19株、B型2株，亲缘关系较近。本次实验分离的沙门氏菌血清型以7∶c∶5为主，含有16个主要的毒力基因，A型为优势基因型。

对2015年上海地区猪屠宰场、猪养殖场、临床病死猪开展猪链球菌的分离检测,主要包括对猪唾液、猪鼻拭子、猪场空气样本、扁桃体、肺脏等共363份进行细菌分离和PCR鉴定,共分离到猪链球菌43株,对这43株细菌开展mrp、orf2、sly、gdh、gapdh、epf、fbps 7种主要毒力因子检测,通过对病原的分析,了解上海地区猪链球菌病的疫情动态和趋势,为防控猪链球菌病提供依据.

In order to investigate the antibiotic resistance of Escherichia coli of animal origin in Shanghai, antimicrobial susceptibility test of 236 E.coli isolates from animal clinics to 14 antimicrobial agents was surveyed. Among them,164 strains were identified as swine origin,72 strains were identified as avian origin. Broth microdilution method according to the recommendation of the CLSI was used. The results showed that both E. coli from swine and avian showed antibiotic resistance to most antimicrobial agents. E.coli strains were highly resistant to sulfisoxazole,sulfamethoxazole,enrofloxacin and spectinomycin,etc. Lower antibiotic resistance rate( ≤ 20%) against amikacin and amoxicillin or clavulanate was showed. In addition,E.coli from swine had a more complex type of drug resistance spectrum than E.coli from avian.