Preliminary Study On The Patho-ecology Of Two Emerging Diseases Of Shrimp

Viral covert mortality disease(VCMD)and acute hepatopancreatic necrosis disease(AHPND)were two emerging diseases that threatened the shrimp of aquaculture in recent years.The spread of these two diseases caused severe economic losses of shrimp farming industry in China.Studies had confirmed that the covert death nodavirus(CMNV)and the various Vibrio carrying the plasmids with Pir genes were the causative agents of VCMD and AHPND,respectively.Based on the systematic study of the CMNV infection in the four main cultured shrimps species in China,including Penaeus vannamei,Penaeus monodon,Penaeus japonicus and Macrobrachium rosenbergii,further investigation of CMNV infection in the co-habiting organisms in/around the shrimp farming ponds and the Portunus pelagicusthan collecting from the East China sea were conducted by using the techniques of molecular biology,histology and ultrastructural pathology to explore whether these species were the reservoirs of CMNV.Meanwhile,this study also attempted to conduct a preliminary study on the patho-ecology of AHPND-causing Vibrio parahaemolyticus(VpAHPND).The infection of VpAHPND in the Portunus trituberculatus were investigateded by using the methods of nested PCR,histopathological section and in situ hybridization.The reservoir host and vectors of VpAHPND in the shrimp culture pond and surrounding environment was also inspected in the presenct study.In the investigation of the CMNV prevalence in farming shrimp,the result of reverse transcription nested PCR assay showed that the total RNA from samples of P.vannamei and P.monodon were able to produce the amplicons of expected size.The sequencing results of the amplicons confirmed that the amplified fragment was the CMNV gene.The analysis of phylogenetic tree based on the sequence of the sequenced fragment also showed that the CMNV strains in the two shrimps were closely related to the original CMNV strain.Histopathology,in situ hybridization(ISH)and transmission electron microscopy(TEM)analysis of the diseased P.vannamei,P.monodon,P.japonicus and M.rosenbergii revealed the presence of nuclear pyknosis and necrosis in the hepatopancreatic tubular epithelial cells of these shrimp samples.Muscle tissues of P.vannamei and M.rosenbergii showed dissolving necrosis and loosening of muscle fibers.The positive hybridization signal of CMNV RNA probe was present in the lesions of hepatopancreas and muscles.The TEM results confirmed the presence of a large amount of scattered CMNV-like virus particles in the hepatopancreatic tissue of these samples.The above results indicated that the diseased individuals of the four major species of farming shrimp were infected by CMNV and the infection caused severe pathological damage in hepatopancreas and muscle tissue of these diseased individuals.In the investigation of CMNV carrying and infection in wild jellyfish from shrimp culture ponds and surrounding drainage channels,the results of reverse transcription nested PCR amplification showed that expectant CMNV bands could be amplified from the total RNA of the Gonionemus vertens samples,no expectant CMNV bands were amplified in the Aurelia aurita samples.The positive rate of CMNV in 32 individuals of G.vertens was 12.5%.The sequencing of expectant CMNV bands and sequence alignment showed that the CMNV target sequences from the G.vertens shared 99%similarity with that form the original CMNV strains.The phylogenetic tree analysis showed that the CMNV sequence from the G.vertens and original CMNV strains were clustered tightly together.Histopathological analysis showed that pathological damage occurred in the stomach,muscle and other tissues of the G.vertens.ISH analysis revealed that obvious positive hybridization signals of CMNV RNA probes were presented in the lesions.TEM analysis confirmed the presence of a large number of CMNV-like virus particles in the tissues of the G.vertens stomach and muscle.The result of CMNV naturally infection in the jellyfish further confirmed the ability of cross-species transmission possessed by CMNV.From 2017 to 2018,the prevalence of CMNV in co-habiting organisms in shrimp culture ponds in coastal provinces of China and the infection in the samples of Portunus pelagicus collected from the East China Sea were investigated.A total of 100 samples including 34 different species were collected.The analysis results of these samples showed that CMNV-positive samples included thirteen species including the Juvenile of P.vannamei,Caridina cantonensis,M.rosenbergii,Sinocorophium sinensis,Neomysis awatschensis,Helice tridens tientsinensis Rathbun,Portunus trituber culatus,Oncomelania hupensis Gred1er,Brine Shrimp,Musca domestia,Carassius auratus,Mugilogobius abei and Oreochromis mossambicus.The prevalence rates of CMNV were 40.9%and 26.8%in the co-habiting organisms of shrimp farming ponds in coastal areas culture ponds in 2017 and 2018,respectively.Reverse transcription nested PCR detection and target gene sequencing revealed that expectant CMNV fragments were amplified in samples such as Helice tridens tientsinensis Rathbun,Ocypode cordimand,Neomysis aoatschensis,Corophium,Mugilogobius abei and Musca domestia.The sequences from the co-habiting organisms shared 97%similarity with that of original CMNV strain.The results of phylogenetic tree analysis showed that the sequence of CMNV from the co-habiting organism and original CMNV strains were clustered tightly together.The pathological analysis showed that pathological changes could be observed in multiple tissues of the co-habiting organism samples.The ISH results showed that blue-violet CMNV probe positive hybridization signal were presented in the lesions of the above samples.The TEM analysis confirmed that there were a large number of CMNV-like virus particles in the juvenile of P.vannamei,H.tridens tientsinensis Rathbun,O.cordimand and N.succinea.The aboving results indicated that CMNV could infect species such as H.tridens tientsinensis Rathbun,O.cordirnand,N.succinea and P.pelagicus under natural conditions.These species might play an import role in in the transmission and epidemic of CMNV.In order to exploring the possible pathogenic agents related to the slow growth of Portunus trituberculatus from the shrimp and crab poly culture ponds in Weifang City,Shandong Province,the present study investigated the status of carrying or infecting of nine pathogens including white spot syndrome virus(WSSV),Infectious Hypodermal and hematopoietic necrosis virus(IHHNV),shrimp hemocyte iridescent virus(SHIV),VpAHPND,enterocytozoon hepatopenaei(EHP),covert mortality nodavirus(CMNV),yellow head virus(YHV)and hepatopancreatic parvovirus(HPV)in the samples of P.trituberculatus by using the molecular biological detection methods.Meanwhile,histopathological analysis and in situ hybridization(ISH)were performed on the samples.Results of molecular biological analysis showed that the P.trituberculatus samples of slow-growing were tested to be positive of VpAHPND by nested PCR,while the other seven pathogens were not detected in the samples.Sequencing and sequence alignment analysis based on the amplicons of VpAHPND nested-PCR of the samples revealed that the amplified product from P.trituberculatus samples shared 99%identity to the pirAvp virulence gene from the pathogenic V.parahaemolyticus plasmid.The analysis of phylogenetic tree indicated that the sequence from P.trituberculatus samples is clustered into the same main branch with the reported pathogenic Vibrio parahaemolyticus PirA.Histopathological analysis revealed necrosis of epithelial cells in hepatopancreas tubules,dissolving myonecrosis of the heart,necrosis of epithelial columnar cells of gills,severe nerve cell damage in the thoracic central ganglion,and nuclear pyknosis in these tissues.The results of ISH showed that blue-violet hybridization signals of the VpAHPND probe were present in the lesions of the hepatopancreas,myocardium,gills,and thoracic central ganglia.The above results indicated that the P.trituberculatus samples from the shrimp and crab polyculture ponds were naturally infected by VpAHPND,which caused serious pathological damages of the hepatopancreas,myocardium,gills,and thoracic central ganglia.The present study revealed,for the first time,that VpAHPND infection in P.trituberculatus and the pathological changes caused by infection.The results provide basic information for revealing the natural host species of VpAHPND,and the prevention and control of AHPND of cultured P.trituberculatus.In order to find out the natural host and vector species of VpAHPND,109 samples including organisms from farming ponds of P.vannamei,M.rosenbergii,P.japonicus,P.trituberculatus,and wildlife species inhabiting around the pond including P.vannamei,H.tridens tientsinensis Rathbun,C.intestinalis,Uca,Copepods and Cladocera,Nereis succinea,Genus Corophium,Calcarina,Brine Shrimp,Balanus were collected.The prevalence of VpAHPND in the co-habitting organisms in 2017 and 2018 was 23.53%and 26.67%,respectively.Molecular biological analysis of nested PCR and real-time PCR showed that some P.vannamei,P.japonicus,P.trituberculatus,H.tridens tientsinensis Rathbun,Copepods and Cladocera,M.domestia were positive for VpAHPND,Ciona intestinalis,Genus Corophium,Ciona intestinalis,Brine Shrimp,Balanus,and Nereis succinea were negative for VpAHPND.The results of ISH analysis confirmed the presence of positive hybridization signals of VpAHPND probe in the hepatopancreas of the H.tridens tientsinensis Rathbun,and muscle tissue of P.vannamei.The above studies indicated that the P.vannamei in the culture pond and H.tridens tientsinensis Rathbun in the drainage channel around the culture pond could be infected by VpAHPND,and the H.tridens tientsinensis Rathbun and clam worm might play a role as a vector in the spread the VpAHPND in the culture pond.The present study indicated that CMNV was prevalent and epidemics in cultured shrimps including P.vannamei,P.monodon,P.japonicus and M.rosenbergii,in many coastal provinces of China,and that various organisms in shrimp culture ponds might be acted as the CMNV reservoirs for its infection,storage and transmission,and that CMNV possessed the capacity of cross-species transmission.These ecological characteristics of CMNV suggested that the risk of VCMD prevenlace in shrimp aquaculture were worthy of attention.Meanwhile,the confirmation of VpAHPND naturally infection in P.trituberculatus and various symbiotic organisms in the shrimp farming ponds demonstrated that it might be not easay to control the prevalence of VpAHPND in the future.The relevant results of this study enriched the patho-ecology ecology of CMNV and VpAHPND on the one hand,and provided theoretical support for the effective prevention and control of these two emerging diseases on the other hand.