随着养鹅业不断走向规模化、交通物流加速和养鹅环境污染,鹅的各种疾病呈现出一些新的特点,如何根据这些特点找到有效减少疾病发生的对策,是养鹅业能否健康发展的重要环节.本文从当前鹅主要流行病的病原学、流行特点、临床症状以及病理变化等方面进行概述,并提出相关防控措施,希望能为鹅病进一步研究和生产有效防控提供理论指导和技术支持.

水禽呼肠孤病毒病是当前主要流行且危害水禽养殖最严重的水禽疫病之一.该病因其病原变异快,感染的宿主谱广,导致宿主免疫抑制,进而诱发细菌的继发感染,使该病呈现出多样化、复杂化的特征.该病不同基因型病毒引起的临床表现变化不大,但引起病理变化差异比较大,可表现为肝脾坏死、肝脾出血、肝脾出血同时伴有坏死等多种不同病变.本文在笔者研究基础上,参考结合其他单位研究成果,对当前水禽呼肠孤病毒病的病原学、流行病学特点、临床症状及病理变化等方面进行概述,并提出相关防控措施,希望能为水禽呼肠孤病毒病进一步研究和有效防控提供理论指导和技术支持.

从广东四会地区某猪场送检的病死猪肠管内容物中分离到1株疑似猪魏氏梭菌的细菌.经形态学观察、培养特性鉴定、生化试验、致病性试验,显示该茵为魏氏梭菌.药敏试验结果表明,该分离菌对庆大霉素、阿米卡星、卡那霉素等高敏,对四环素、氨苄西林、磺胺甲恶唑等耐药.

采用RT-PCR方法对NDRV-QY分离株的S组基因编码区进行RT-PCR扩增、克隆及序列测定.结果表明,QY株S组基因包含的ORF大小分别为1 517 bp、1 251 bp、1 104bp和1 104 bp.将QY株的S组基因各节段编码区核苷酸序列及其推导氨基酸序列与正呼肠孤病毒属的呼肠孤病毒的相似性比较和遗传进化分析结果表明,QY株与ARV、TRV、MDRV、NDRV均有不同程度的同源性,其中与NDRV的同源性最高,核苷酸序列和氨基酸序列同源性均达90％以上.QY株与NDRV、MDRV、ARV同在正呼肠孤病毒的第Ⅱ亚群,与NDRV处在同一个分支,与MDRV和ARV处在不同分支,建议将新型鸭呼肠孤病毒分类在正呼肠孤病毒属的第Ⅱ亚群当中.

To develop a method for simultaneous detection of new—type duck reovirus(NDRV) and muscovy duck reovirus(MDRV),a duplex RT-PCR was established with 2 pairs of specific primers designed based on the conserved sequences in S3 gene of NDRV and MDRV.Two special fragments of 298 bp(NDRV) and 189 bp(MDRV) were amplified by the optimized duplex RT-PCR.This naothed was tested on 42 clinical samples of sick ducks from different areas of Guangdong province,of which 19 were positive for NDRV and 8 were positive for MDRV by either the duplex RT-PCR or single RT-PCR,and could be used in clinical diagnoses.

为建立一种能够快速检测新型鸭呼肠孤病毒(NDRV)的方法,本研究参考GenBank上登录的NDRV S3基因保守序列设计特异性引物.经条件优化后,建立了检测NDRV的RT-PCR方法.对其特异性、敏感性和重复性进行检验.结果显示:该方法仅能从NDRV分离毒中扩增到与预期大小相符,长度为298 bp的特异性目的片段,检测灵敏度达到83.4 pg病毒RNA;而其它病毒:番鸭呼肠孤病毒、禽呼肠孤病毒、鸭病毒性肝炎病毒、鸭瘟病毒、鸭新城疫病毒和禽流感病毒等样品的扩增结果均为阴性.采用该方法对在广东不同地区采集的15份鸭病料样品进行检测,NDRV阳性率为53.33％.表明建立的RT-PCR方法特异性强、敏感度高,可用于NDRV的临床诊断和流行病学调查.

鸭瘟,又名鸭病毒性肠炎,是由疱疹病毒科鸭瘟病毒引起的鸭、鹅和天鹅等水禽的一种急性、热性、败血性传染病.患病鸭表现为高热,两腿麻痹,下痢,流泪;部分病鸭头颈肿大.病变以食道和泄殖腔黏膜出血或有灰黄色假膜覆盖为主要特征[1-2].2014年6月中旬,广州花都炭步镇某养殖户送检病死鸭,经诊断为鸭瘟.现报告如下. 1 饲养和发病情况 广州花都炭步镇某养殖户共饲养2 800只蛋鸭,发病前蛋鸭产蛋率为85％.该养殖户淘汰了500只450日龄蛋鸭后,2014年6月5日从湖南衡阳引入800只80日龄新蛋鸭混养.6月8日鸭群开始发病.

参考GenBank新型鸭呼肠孤病毒(New-type duck reovirus,NDRV)S3基因序列设计合成一对引物,对新型鸭呼肠孤病毒QY株S3基因进行RT-PCR扩增,并对PCR产物进行了克隆和测序.结果显示扩增产物为1 104 bp,与预期的目的片段大小一致.相似性分析QY株S3基因核苷酸序列与ARV代表株、MDRV代表株和DRV代表株,相似性分别59.4％ ～ 60.0％、61.1％～ 61.3％和96.7％～ 98.6％;氨基酸的相似性分别为67.6％～ 68.7％、68.1％～68.9％和95.4％～ 98.4％.表明QY株的S3基因具有不同于ARV和MDRV的特征,分离病毒QY株是不同于禽呼肠孤病毒和番鸭呼肠孤病毒的独立基因群.

T1 strain of Haemophilus parasuis(HPS) was isolated from the heart,liver and lung samples of sick swine which were suspected to be infected with HPS in Foshan,Guangdong province.The T1 strain is identified based on morphologic,cultural,biochemical characteristics and PCR detection of HPS.Drug sensitive test showed that the HPS isolate was highly sensitive to tetracycline and ofloxacin,but was strong drug resistant to oxacillin and sulfamethoxazole.And animal pathogenicity test indicated that the T1 was highly virulence.

A virus was isolated from the liver and spleen of the infected cheeper Muscovy ducks in Guangdong Province.The sick ducks showed weakness in legs and pathological findings included hemorrhage necrosis in the spleen and liver.The virus isolate designated was demonstrated different from those previously documented in pathogenecity in ducks,virus morphology under electron microscopy,SDS-PAGE analysis of the viral genome and genetic analysis by RT-PCR.Thus,the virus isolate designated was a new-type duck reovirus,and was named DRV-QY strains.

In this study, a SYBR Green Ⅰ real-time RT-PCR assay was developed for detection of new-type duck reovirus(NDRV) with the specific primers designed according to the conserved sequence of S3 genes of NDRV in GenBank. The results showed that recombinant plasmids were used to generate standard curve with correlation coefficient(R2) of 0.9996, and the melting curve showed one specific peak. No cross-reaction was detected for other duck viruses and the limit detection of real-time RT-PCR was about 29 copies/μL for the target gene, which was 100 times higher sensitivity than conventional PCR. The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than 2%. The method established in this study could be useful for earlier rapid laboratory diagnosis and quantitative analysis of the infection of NDRV.

Sialyltransferases are the enzymes which catalyze the biosynthesis of the sialic acid,and this process is closely related to host specificity of influenza virus.The gene of α-2,3 sialyltransferase III(ST3Gal III),of Chinese Yellow Chicken was amplified by RT-PCR method with the total RNA extracted from bursa of fabricius tissue.To construct the expression vector pIRES2-EGFP-ST3Gal III,the amplified gene was cloned into the vector pIRES2-EGFP with the reported gene EGFP(enhanced green fluorescence protein).The positive recombinant pIRES2-EGFP-ST3Gal III was transfected into 293 T cells by liposomes.The expression of the recombinant pIRES2-EGFP-ST3Gal III in the 293 T cells was observed by fluorescence microscope and tested by Western blot.The result indicated that ST3GalIII was successfully expressed in the 293 T cells.

Newcastle disease virus strain GD1002,isolated from the diseased ducks in Guangdong province in 2010,was velogenic according to the pathogenetic tests and highly pathogenicity to duck.The results of virulence determination showed that the MDT,ICPI and IVPI were 56.0h,1.64and 2.29,respectively,belonged to velogenic strain virulence criterion.The Fgene of GD1002strain was amplified by reverse transcription polymerase chain reaction(RT-PCR).The cleavage motif 112 RR-Q-R-R-F117of F protein was identical to the characteristic of velogenic strain.For genotyping,phylogenetic tree based on the nucleotide 47to 420nt of the Fgene were performed using the isolate and other NDV sequences obtained from GenBank.The results revealed that the isolate belonged to genotypeⅨ.And the results of nucleotide sequence homology and evolutionary processes analyses showed that the isolate had a close relationship with F48E9,JS/1/97/Ch and FJ/1/85/Ch strains.

To prepare efficient subunit vaccines against porcine circovirus type 2(PCV2),a sequence encoding multi-epitope of PCV2 was artificially synthesized and ligated into pET32a(+) plasmid.The resultant plasmid was transformed into E.coli BL21 in which a fusion protein was expressed with IPTG induction.The SDS-PAGE analysis showed that fusion protein was approximately 27 ku,mainly in form of inclusion body.Western blot and ELISA detection indicated that the fusion protein reacted with positive PCV2 antibody.This study provided an alternative way to develop a potential PCV2 subunit vaccine and diagnostic reagents.