Pathological Changes in Andrias davidianus Infected with Chinese Giant Salamander Ranavirus

Chinese giant salamander ranavirus （CGSRV） is an emerging pathogen in captive populations of the Chinese giant salamander （Andrias davidianus）. We processed 140 morbid Chinese giant salamanders from seven captive breeding populations over five years, and describe the disease associated with CGSRV infection. The most common gross signs were significant swelling of the legs and coelomic cavity, erythema of the legs and ventrum in juveniles; cutaneous erosions and ulcerations in adults, particularly the limbs and the head; and marked petechial or ecchymotic hemorrhages of the internal organs, particularly the liver, spleen and kidney. Histological examination showed degeneration, necrosis, and inflammation in many organs, particularly in the organs where hemorrhage was observed. There was evidence of eosinophilic inclusion bodies in degenerated and necrotic cells. We identified virus particles and empty capsids without viral nucleoid in the inclusion bodies using electron microscopy. Virus particles were hexagonal or round shape, and appeared in paracrystalline arrays, aggregates, or singly. All enveloped viral particles were 140-160 nm. Polymerase chain reaction followed by sequencing verified that the virus particles were CGSRV. These results collectively support that CGSRV was the etiologic agent responsible for these mass die-offs of the Chinese giant salamander. The pathology described herein will be useful in diagnosing cases of ranaviral disease caused by CGSRV, and provide evidence that this pathogen is a significant threat to the Chinese giant salamander.

The omp2 Gene of HPS-type Bacteria Cloning and Sequence Analysis Isolates from Sichuan Province

In order to compare the homology and antigen of Haemophilus parasuis (HPS)4 outer membrane protein P2(omp2), we design a test with specific primers, using PCR amplification of isolates of Haemophilus parasuis from Sichuan Province(HP Sch2010), ompP2 gene will be cloned into the pGM-T vector, and transformed into E. coli DH5α. Identified by PCR and sequencing and analysis, the sequencing results showed that the published 4 HPS SW124 strains omp2 gene (1077bp), compared with the amplified 1086bp purpose fragment(containing omp2 genes), is relatively stable, with the nucleotide homology level 97% and amino acid homology level of 92.5%. The variable regions are mainly concentrated in the three base sequences: 40-65,110-156,180-202.

兔出血症病毒2型理化特性研究

【目的】本研究旨在研究该病毒的理化特性,评价不同处理条件对RHDV2的杀灭效果。【方法】本研究拟对临床疑似RHDV2感染致死的家兔进行RT-PCR鉴定病原,并利用不同pH值、不同温度、常用兽用消毒剂、不同浓度甲醛处理RHDV2,通过PMA-RT-qPCR对病毒理化特性进行研究。【结果】经RT-PCR检测与测序分析,确诊为RHDV2感染,该毒株VP60基因序列与GenBank中RHDV2(登录号:MN276176.1)一致性为98.35%。PMA-RT-qPCR结果表明,RHDV2对酸碱、高温、兽用消毒剂、甲醛均表现不同程度的敏感性。酸碱处理病毒,随pH值降低或升高,病毒杀灭率均有提高。65℃处理病毒30 min后,病毒杀灭率可达77.61%,83℃处理5 min后,病毒杀灭率可达95.10%。RHDV2对3种所测兽用消毒剂均敏感,其中聚维酮碘对RHDV2杀灭作用最好,作用30 min后,病毒杀灭率达88.78%。0.3%甲醛的杀灭率优于0.2%甲醛,作用30 min后,其杀灭率可达82.22%。【结论】本研究探究了RHDV2的理化特性,该病毒对酸碱、高温、兽用消毒剂、甲醛均有不同程度的敏感性。本研究为RHDV2的诊断、临床消毒剂的选择提供了参考依据,为该病毒致病机理研究与疫苗研发奠定基础。

一株林麝肺源创口博德特氏杆菌全基因组及细胞致死性肿胀毒素分析

【目的】为探究林麝的死亡原因,本研究无菌采集1只死亡林麝肺脏后进行细菌的分离鉴定。【方法】通过细菌分离纯化、生化试验和16S rRNA序列分析,对分离菌株进行鉴定;然后通过药敏试验、全基因组测序与分析以及细胞致死性肿胀毒素(cytolethal distendin toxin subunit B,CDTB)分析,对分离菌株耐药性和致病性进行研究。【结果】在死亡林麝肺脏中分离出1株革兰阴性菌,经鉴定为创口博德特氏杆菌,命名为ZL0001。经过细胞培养观察和CDTB分析表明该菌为胞内寄生菌,含有细胞致死性肿胀毒素,能导致细胞凋亡。药敏结果表明该分离株对氨苄西林、亚胺培南、链霉素和四环素等药物敏感;对氨曲南、林可霉素、克林霉素和呋喃妥因耐药。全基因组测序分析结果表明,该菌株与其他创口博德特氏杆菌的平均核苷酸一致性(average nucleotide identity,ANI)值均>97%,基因组大小为4350644 bp,共发现22个耐药相关基因。此外,该菌株基因组包含63个毒力相关基因,其参与鞭毛蛋白、脂多糖、铁摄取、抗血清蛋白以及细胞致死性肿胀毒素等毒力因子的合成。【结论】本研究首次在动物呼吸道中分离出创口博德特氏杆菌,并证明其为胞内寄生菌,对林麝肺炎的发病机理研究具有重要意义。