四川地区一株传染性造血器官坏死病毒的分离鉴定及系统发育分析

2014年5月,四川省都江堰市某虹鳟养殖场暴发一种传染性疾病,幼鱼和鱼苗死亡率分别高达40％和80％.为探究此次疾病病因和流行规律,将病料进行解剖及细菌学检查、病理组织观察、人工感染实验、病毒分离、多重RT-PCR鉴定和系统发育分析.结果显示,病鱼主要临床症状表现为腹部膨大,体表发黑,肛门拖淡黄色黏液便,解剖见鳔壁、腹膜出血,胃胀气膨大和明显肠炎;细菌学检查为阴性;组织病理学上,头肾、肾脏和脾脏造血组织广泛性变性、坏死,肠黏膜下层嗜酸性颗粒细胞浸润与坏死,肝细胞变性、坏死形成局灶性的坏死灶,并在一些肝细胞胞浆内见嗜酸性包涵体.将病鱼的肝脏、脾组织研磨过滤灭菌后,腹腔注射20尾健康虹鳟,注射组均表现为急性死亡(累积死亡率=75％),并出现与自然发病鱼相同的症状.取病鱼组织匀浆滤菌液接种到鲤上皮瘤细胞(epithelioma papulosum cyprini,EPC),盲传3代出现典型的细胞病变效应(cytopathic effects,CPE).针对IHNV、IPNV与VHSV的多重RT-PCR检测显示自然发病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV核蛋白(nucleoprotein,N)基因同源性为99.9％.对分离株的糖蛋白基因“Mid-G”区域进行系统发育分析,结果显示该分离株与亚洲分离株聚为一支,属于JRt基因型.本研究首次报道我国西南地区养殖虹鳟中IHNV感染引起的疾病.     

四川彭州地区养殖虹鳟传染性造血器官坏死病病毒的分离、鉴定及病理学观察

近期,四川省成都彭州市某虹鳟养殖场暴发流行疾病,导致养殖虹鳟死亡率高达90%。现场采样观察发现患病鱼主要症状为背部发黑,鳔壁、腹膜严重出血,心包积液,空肠、空胃和显著肠炎。同时对病鱼进行细菌学与组织病理学检测,细菌学检查结果为阴性,病理学观察发现脾脏有典型凝固性坏死,肝脏组织广泛变性、坏死,肠道黏膜下层水肿,肠上皮充血及上皮细胞脱落坏死,脑膜和心外膜水肿。将病鱼的脾组织研磨过滤除菌后,腹腔注射60尾健康虹鳟,注射组均表现为急性死亡(累计死亡率达85%),试验鱼出现与自然发病鱼相同的症状而对照组无异常。取病鱼的脾脏组织研磨过滤后接种胖头鲤细胞(fathead minnow cell,FHM),细胞感染3 d后出现典型的细胞病变效应(CPE)。针对编码IHNV糖蛋白(Glycoprotein,G)基因进行逆转录-聚合酶链反应(RTPCR)检测显示,患病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV糖蛋白基因同源性为98.2%。对该病毒分离株的G基因进行系统发育分析,结果显示,该分离株与亚洲分离株聚为一簇,属于JRt基因型。     

传染性造血器官坏死病毒新疆株的分离与鉴定

在新疆维吾尔自治区某养殖场取患病虹鳟Oncorhynchus mykiss组织样本,进行传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)分离鉴定、电镜观察、回归感染及遗传进化分析研究。细胞培养结果显示:患病虹鳟组织样本能够感染鲤Cyprinus carpio上皮细胞(carp epithelial cell,EPC)产生典型细胞病变(cytopathic effect,CPE),收集病毒悬液命名为XJ-13。滴度测定实验表明:该病毒为10~(6.15)TCID_(50)/m L。回归感染实验表明:该分离株在浓度为10~5PFU/尾的剂量使体质量5g的虹鳟死亡率达87.5%。通过透射电镜观察发现患病虹鳟组织悬液感染的EPC细胞内存在大量的子弹状病毒粒子,PCR鉴定结果表明该病毒为IHNV。XJ-13的糖蛋白氨基酸序列的聚类分析结果显示,该病毒株与我国IHNV-Sn1203株具有最高的同源性(99%),与美国参考株WRAC的同源性为94.6%。结果表明:IHNV XJ-13是造成该养殖场虹鳟大量死亡的病原。     

传染性胰腺坏死病病毒分离株VP2基因抗原表位区融合表达及免疫特性的分析

利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

一株鲈鲤源鲤春病毒血症病毒的分离鉴定及其病理观察

2016年4月,四川某鲈鲤(Percocypris pingi)养殖场流行一种以鳃、鱼鳔和内脏器官出血为临床特征的传染病。组织病理学观察发现,患病鲈鲤全身多组织器官均发生明显的病理损伤,尤其是肝、脾、肾、鳃和肠表现为明显的出血、变性、坏死以及炎症细胞浸润。取病鱼组织匀浆滤液接种鲤上皮瘤细胞(epithelioma papulosum cyprini, EPC),盲传3代出现典型的细胞病变(cytopathic effects, CPE)。将自然发病鱼组织匀浆滤液和细胞培养病毒液分别接种健康鲈鲤,实验鱼出现与自然发病鱼相同的症状,死亡率分别为60%和50%,而对照组未见异常。对经分离毒株ZLP160415感染出现CPE的EPC细胞制备超薄切片进行电镜观察,发现病毒呈弹状,长90～150 nm,宽40～60 nm;对自然发病鱼、人工感染发病鱼内脏组织和细胞培养病毒液进行鲤春病毒血症病毒(springviremiaofcarpvirus,SVCV)的RT-PCR检测,均扩增出目的条带。基于SVCV糖蛋白基因进行系统发育分析,结果显示分离毒株(ZLP160415)属于Ia型。结合本次疾病的流行病学与病理损伤特点、病毒分离鉴定、人工感染试验结果和透射电镜检查,确定此次流行病的病原为SVCV。     

一株似鲇高原鳅源蛙病毒的分离与鉴定

从四川乐山某养殖场自然发病的似鲇高原鳅(Triplophysa siluroides)体内分离到一株病毒FYL140220。病毒感染鲤上皮瘤细胞系(epithelima popuasum cuprini,EPC)后,细胞呈现圆缩、坏死、脱落、明显空斑的病变特征。将自然发病鱼组织过滤除菌液和细胞培养病毒液分别接种健康似鲇高原鳅,试验鱼出现与自然发病鱼相同的症状,死亡率分别为30%和40%,而对照组无异常。对经FYL140220感染出现典型细胞病变(CPE)的EPC细胞制备超薄切片进行电镜观察,发现病毒呈正六边形,对称20面体,对角线直径约(103±7)nm;提取细胞培养病毒液、自然发病鱼和人工感染发病鱼的内脏器官总DNA进行蛙病毒主要衣壳蛋白(maior capsid protein,MCP)基因保守区域的PCR扩增,均能扩增出预期大小约500 bp的特异性条带。MCP基因同源性与遗传进化分析表明,分离株FYL140220与蛙病毒属成员聚为一支,尤其与大鲵蛙病毒与沼泽绿蛙病毒同源性最高,分别达99.8%和99.6%。结合PCR检测、电镜观察和系统发育分析,确认分离病毒FYL140220为蛙病毒,这是首次报道蛙病毒可自然感染似鲇高原鳅并致死。     

斜带石斑鱼溶藻弧菌病的研究

从患病的斜带石斑鱼肝和肾组织分离的两株病原菌,进行纯化培养、人工感染、VITEK-AMS-32自动微生物分析鉴定及药敏试验,经形态和生理生化测定,确定为溶藻弧菌(Vibrioalginolyticus)。组织病理变化主要表现为鳃、肝和肾细胞变性、坏死,病变组织炎性细胞浸润,呈变质性炎症。     

四川地区养殖鲫鲤疱疹病毒Ⅱ型的鉴定及病理学研究

为探究疾病病因和流行特点,对患病鲫进行剖检、病理学观察、分子生物学检测及人工感染实验。结果显示,患病鲫临床表现为离群独游、浮头、全身发黑及体表出血等。剖检发现鳃、肝脏、肾脏等器官出血、肿胀和坏死,鳔点状出血。组织病理观察显示,鳃呼吸上皮细胞肿胀、坏死及脱落;肾脏局灶性坏死;脾脏组织广泛变性坏死,造血系统崩解;肝脏发生空泡变性和坏死。透射电镜观察到脾脏和肾脏中存在4种大小不同的病毒颗粒,分别为DNA病毒内核、空衣壳、实心衣壳和有包膜的成熟病毒粒子。未成熟的病毒粒子存在于细胞核,成熟的病毒粒子存在于细胞质。电镜下可见鲤疱疹病毒Ⅱ型(CyHV-2)在细胞核内完成核酸复制和核衣壳装配,囊膜蛋白在出核后获得。细胞核萎缩、核染色质边缘化,线粒体肿胀、坏死、嵴断裂,细胞质空泡化。取患病鲫组织匀浆滤菌后接种到鲤上皮瘤细胞系(epithelioma papulosum cyprinid cell line,EPC)和胖头鲤肌肉细胞系(fathead minnow cell line, FHM),盲传3代后未见细胞病变。通过腹腔注射患病鲫组织匀浆进行人工感染实验,实验组鲫表现出与自然感染鲫相同的临床症状,对照组鲫正常,实验组累积死亡率为80%。用PCR方法检测CyHV-2的解旋酶基因片段,扩增出预期大小相符的目的条带。利用邻近法对该病毒的解旋酶基因进行系统发育分析,结果显示与CyHV-2(YZ-01)的同源性为99%。以上实验结果证实了CyHV-2是导致这次疾病暴发的原因。     

虹鳟源嗜冷黄杆菌的分离鉴定及致病性

为确定患病虹鳟的病原,本实验从患病鱼溃烂肌肉中分离到2株细菌,分别命名为CH06和CH07,经回归感染证实分离菌株为导致此次虹鳟患病的病原菌,并进一步对其形态特征、理化特性、分子特征、血清型及耐药性进行分析。结果显示,CH06和CH07株在TYES琼脂平板上呈煎蛋状外观,产黄色素,氧化酶和过氧化氢酶呈阳性,能水解明胶和酪蛋白,不能水解淀粉,不能利用果糖、半乳糖和七叶苷等。16S rRNA比对结果显示,CH06和CH07株与嗜冷黄杆菌模式株NBRC 15942的同源性分别为99.35%和99.42%。综合菌株理化和分子特性确定CH06和CH07株为嗜冷黄杆菌。利用多重PCR方法鉴定CH06和CH07株的血清型均为1型(Fd型);多位点序列分型(MLST)分析表明,CH06和CH07株的基因型分别为ST-12和ST-78型,且均属于CCST10克隆型。人工感染结果显示,CH06和CH07株对虹鳟幼鱼具有较高致病性,其半致死浓度(LD_(50))分别为7.1×10~5和1.1×10~5 CFU/mL,攻毒剂量与临床病症出现时间呈反比,从人工感染实验鱼的肌肉、脾脏等组织中可重新分离到嗜冷黄杆菌。组织病理变化显示,病鱼肝细胞肿胀,空泡变性,部分肝细胞溶解坏死,细胞核溶解消失;脾脏充血、出血,淋巴细胞减少,红细胞和含铁血黄素增多;肌纤维间隙增宽、断裂、弯曲不齐,部分肌细胞肌浆溶解呈蜂窝状。CH06和CH07株对10种抗菌药物的耐药谱略有不同,均对氨苄西林和甲氧苄啶-磺胺甲噁唑敏感;CH06株对恩诺沙星、氟苯尼考等耐药,而CH07株对恩诺沙星和氟苯尼考中度敏感。本研究首次报道了我国虹鳟源嗜冷黄杆菌的分离鉴定及生物学特性,以期为虹鳟细菌性冷水病的防控提供科学依据。     

Infectious pancreatic necrosis virus (IPNV) serotype Sp is prevalent in Turkish rainbow trout farms

Infectious pancreatic necrosis virus (IPNV) is a common pathogen of rainbow trout (Oncorhynchus mykiss) in Turkey. We found that 455 of 1,676 sample pools tested were IPNV positive. Positive samples were found in all geographical regions where sampling was conducted. Sequence and phylogenetic analyses of VP2 from 30 isolates representing all regions showed that the viruses were highly similar in sequence and grouped within Genogroup 5 (serotype Sp‐A2). No correlations between sequences, sampling sites or geographical origins were identified. Although clinical disease was evident in several farms, analyses of the amino acid sequence of VP2 showed that all virus strains harboured the P₂₁₇T₂₂₁ motif, assumed to be associated with low virulence. We conclude that IPNV is prevalent in Turkish rainbow trout farms and that the viruses are very homogenous and likely to be of European origin. Frequent exchange of eggs and live fish within the farming industry may explain the homogeneity of the IPNV.     

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.     

Tissue distribution of infectious pancreatic necrosis virus serotype Sp in naturally infected cultured rainbow trout,Oncorhynchus mykiss (Walbaum): an immunohistochemical and nested‐PCR study

This study investigates the occurrence and distribution pattern of infectious pancreatic necrosis virus (IPNV) within the pancreas, liver, kidney and spleen of naturally infected cultured rainbow trout, Oncorhynchus mykiss (Walbaum), using immunohistochemistry (IHC). A nested PCR was also employed to confirm the presence of the virus in the pooled tissues of the specimens. All the examined tissues except spleen were immunohistochemically positive for IPNV, but staining intensity and distribution pattern varied. The kidney tubules had the most intense and widespread staining by IHC, indicating a specific tissue tropism at least for this particular serotype. The nucleotide sequence had the greatest identity with the Sp serotype confirming the presence of the nucleic acid of IPNV in the pooled tissues. Based on the present findings, it could be concluded that the absence of lesions consistent with infectious pancreatic necrosis (IPN) disease in the H&E‐stained sections cannot rule out the presence of the IPNV, and the use of an alternative rapid confirmatory method such as IHC with formalin‐fixed, paraffin‐embedded tissue sections is helpful for the final diagnosis of IPN in rainbow trout.     

Comparison of mortality and viral load in rainbow trout (Oncorhynchus mykiss) infected with infectious pancreatic necrosis virus (IPNV) genogroups 1 and 5

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.