Identification And Characterization Of The Causative Agent Of The Diseases Outbreak In Crayfish Procambarus Clarkii China

The red swamp crayfish Procambarus clarkii,is one of the most important cultured aquatic crustacean,with promising results and huge economic value.It was introduced to Nanjing China from Japan in 1920s,and cultured in all forms of freshwater,in most provinces in China.Disease resistance,high fecundity,high growth rate,short maturation time,strong adaptability to new environment make its distribution easily successful.High market demand and high economic value of this species has increases farmers preference.In May 2018 there was a serious diseases outbreak in the farm in Xinglong Township,Xuyi County,Jiangsu province China,and some of crayfish die in the farm,though,the causative agent was not clearly known.Therefore,this study was conducted to find out and identify the pathogen responsible for the outbreak in crayfish farm.This study employs PCR method to detect and identify the pathogens in the liver of crayfish,and TEM technique to study morphology of parasite in the gills of ill crayfish.The PCR detect two different band of viruses,200bp band,of WSSV,and 150 bp band,of unknown virus in the liver of ill crayfish.The viruses were then confirmed by studding their morphology using TEM.The TEM confirm the present of large number of two different viral particles in the gills of ill crayfish.The virus particles differ in morphology and size.One particle was rod shaped with envelop surrounding,of approximately 300nm long,identified as White Spot Syndrome Virus WSSV.This virus WSSV,is well known to cause diseases in crustacean including crayfish Procambarus clarkii.Another virus was spherical to round in shape,non envelop,very small in size,of approximately 30nm diameter,identified as a member of picornavirus.This is a new pathogen in this crayfish Procambarus clarkii.We assign the name for this new picornavirus as Procambarus clarkii picornavirus,PcPV.High throughput sequencing was used to determine the genetic sequence of this new virus PcPV using well designated primers,forward primer:5’-GCAAGGAGCCCGCCACTATT-3’,and Reverse primer:5’-ACATTGCGAGCTGCTTCGTTC-3’.The obtained genetic sequence of this novel virus PcPV,was deposited to GenBank and compared with other picornavirus,by Blast X.The novel virus PcPV,is 75%identical and closest to unclassified Beihai picorna like virus 105 strain BHHK79817.The neighbor-joining tree was constructed from multiple sequence alignments,within a total of 21 genomic sequences of Picornavirales derived from the GenBank database,using molecular evolutionary genetics analysis(MEGA)software,version 6.0,and conserved protein domain was analyzed by searching SMART database(http://smart.embl-heidelberg.de/)and using MOTIF search tool(http://www.genome.jp/tools/motif/),the new virus PcPV is closest and monophylyly with Beihai picorna like 105 strain.The present study suggest that,the novel Procambarus clarkii picorna-like virus PcPV,and Beihai picorna-like virus 105 strain,may possibly present another new genus of family Picornaviridae.This study found that,100%of ill crayfish from the farm were PcPV positive,while only 62.5%of ill crayfish(15/24)were WSSV positive,and 37.5%(9/24)of ill crayfish from the farm were WSSV negative.Also 100%of healthy crayfish from Nanquan market were PcPV negative;however,75%of healthy crayfish were WSSV positive.We concluded that,the disease outbreak from the farm was not only because of WSSV but the novel picorn-like virus PcPV,was to large extent contributed to the outbreak.The novel Procambarus clarkii picorna like virus,PcPV,is a new pathogen of crayfish Procambaraus clarkii.More research work is needed to study the histopathologic effects and mortality of PcPV in Procambaru sclarkii.