Kathy F.J. Tang

Frozen Commodity Shrimp: Potential Avenue for Introduction of White Spot Syndrome Virus and Yellow Head Virus

Abstract Since 1992, white spot syndrome virus (WSSV) and yellow head virus (YHV) have caused mortalities in cultured shrimp throughout Asia. By 1995, WSSV was detected in Texas and South Carolina, and the virus has also been recently reported in Central and South America (Nicaragua, Honduras, Guatemala, Panama, Colombia, Peru, and Ecuador). The importation of live infected shrimp is the principal mechanism by which exotic viruses may be introduced to new geographic regions. However, another probable mechanism is via the importation of infected commodity shrimp from regions where the pathogens are enzootic. Ten different lots of imported frozen tails of Penaeus monodon were screened for WSSV and YHV by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis. In 8 of 10 samples tested, WSSV was detected, and YHV was found in 3 out of the 10 samples. Six of the 10 sample sets of frozen shrimp gave strong positive tests for WSSV or YHV by PCR, and these were selected for bioassay with spe...

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13‑028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.

Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction

Abstract A competitive polymerase chain reaction (PCR) method for quantification of white spot syndrome virus (WSSV) genome was developed. A pair of WSSV primers, designated WSSV341F/R, was selected to amplify a 341-bp DNA fragment from the WSSV genome. For a competitive internal standard, we constructed and cloned a 289-bp DNA fragment, the result of a 52-bp deletion from the 341-bp amplicon. In a competitive PCR reaction, we co-amplified the target WSSV DNA with known concentrations of the internal standard using WSSV341F/R primers. The amplicons from WSSV DNA and from internal standard DNA differed in size and could be distinguished after gel electrophoresis. The concentration of WSSV genomes was determined from its relation to the concentration of the internal standard. The log–log plot of the ratio of the amplicons (internal standard: WSSV) on the internal standard concentration was linear. Using this competitive PCR procedure, we quantified WSSV DNA in the samples of hemolymph and tissues of the cephalothorax of individual WSSV-infected shrimp. The number of WSSV genomes in both hemolymph and tissues corresponded to the severity of infection determined by histological evaluation. In addition, the changes in number of WSSV genomes in the hemolymph during the course of the infection were determined.

Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection.

Abstract A susceptibility study of postlarvae (PL) and juvenile Super Shrimp®, a selected line of Penaeus stylirostris , was conducted to compare their resistance to infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection to that of a specific pathogen free (SPF) population of P. vannamei . Super Shrimp® PLs were fed with IHHNV-infected shrimp tissue for 2 days and then maintained on a pelletized ration for an additional 28 days. PLs were sampled at days 0, 1, 2, 3, 4, 6, 10, 15, 20, 25 and 30. There was no apparent mortality during the experimental period. Tissue DNA extracted from the PLs was analyzed for the presence of IHHNV by PCR. Low levels of IHHNV were detected only in DNA extracts from samples at days 1, 2, and 3. No IHHNV DNA was detected from days 4 to 30. The days that the PLs were weakly IHHNV-PCR positive were during the period that they were being fed with IHHNV-tissue, and thus, the IHHNV DNA signal was suspected to be from the infected tissue used as a feed. Through both histology and in situ hybridization, we confirmed that tissues of Super Shrimp® PLs were not infected with IHHNV. PCR results of another IHHNV challenge study with juveniles of Super Shrimp® were similar to those with PLs. These results indicate that IHHNV did not replicate in the PL and juvenile Super Shrimp®. In contrast, P. vannamei juveniles, which were used as a positive control, showed a more intense IHHNV infection, as determined by PCR detection, beginning at 6 days postchallenge and increasing throughout the remainder of the study. In addition, the IHHNV-infected P. vannamei at 30 days postchallenge showed histological changes characteristic of IHHNV infection and had a positive reaction for IHHNV with in situ hybridization. Our studies show that Super Shrimp® are resistant to IHHNV infection. This is the first unequivocal demonstration of resistance to viral infection in shrimp.

Induced resistance to white spot syndrome virus infection in Penaeus stylirostris through pre-infection with infectious hypodermal and hematopoietic necrosis virus—a preliminary study

Abstract White spot syndrome virus (WSSV) is highly virulent to penaeid shrimp and has been responsible for serious economic losses on shrimp farms throughout the world. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a small DNA virus that also infects penaeid shrimp; and, although once virulent to culture stocks of Penaeus stylirostris , it has not been associated with mass mortalities in recent years. Through three laboratory challenge studies, we discovered an interference interaction between these two viruses in P. stylirostris. In the first bioassay, juvenile P. stylirostris were infected with IHHNV by feeding them with IHHNV-infected tissue. These shrimp, along with a group of non-infected control shrimp, were then fed WSSV-infected tissue. Two days after the WSSV challenge, mortalities began to occur. All of the control shrimp, which were not exposed to IHHNV, died within 5 days. In contrast, replicated challenge groups of the IHHNV pre-infected shrimp had 31% and 44% survival. Quantitation by real-time PCR determined that the surviving shrimp had high levels (10 9 copies per μg DNA) of IHHNV and very low levels (50–400 copies per μg DNA) of WSSV DNA. Moribund shrimp had high levels (10 6 –10 7 copies per μg DNA) of WSSV and low levels (10 4 –10 6 copies per μg DNA) of IHHNV DNA. A second challenge study with P. stylirostris that were heavily infected with IHHNV showed 21 of 23 (91%) shrimp survived 19 days after exposure to WSSV. Ten of these surviving shrimp were re-exposed to WSSV-infected tissue, and six were still alive after 3 weeks. The third challenge study showed that 28% of the IHHNV pre-infected P. stylirostris survived 22 days after exposure to WSSV. In situ hybridization analysis in all the challenge studies confirmed that the surviving shrimp were strongly infected with IHHNV and had, at most, only low levels of WSSV infection. Laboratory WSSV challenges showed that there were no survivals in either IHHNV pre-infected Penaeus vannamei or in IHHNV-resistant P. stylirostris (SuperShrimp™). In conclusion, juvenile P. stylirostris that are highly infected with IHHNV show resistance to WSSV infection.

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries. Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp

A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.

In situ detection of Australian gill-associated virus with a yellow head virus gene probe

Abstract A digoxigenin-labeled gene probe for yellow head virus (YHV) was used to detect gill-associated virus (GAV) in Penaeus monodon from Australia via in situ hybridization. In GAV-infected shrimp, positively reacting tissues included: lymphoid organ, gills, antennal gland, and cuticular epithelium of the stomach. This demonstrates that a YHV probe can be used as a diagnostic tool for GAV and supports previous suggestions that these two viruses are closely related.

Nucleotide sequence of a Madagascar hepatopancreatic parvovirus (HPV) and comparison of genetic variation among geographic isolates

A segment of Madagascar hepatopancreatic parvovirus (HPV) genomic sequence (5742 nucleotides) was determined through PCR and direct sequencing. This nucleotide sequence was compared to isolates from Australia, Thailand, Korea, and Tanzania, and the mean distance was determined to be 17%. The Madagascar HPV is closest to the Tanzania isolate (12%), followed by isolates from Korea (15%), Australia (17%) and Thailand (20%). Analysis of the genomic structure revealed that this HPV sequence is comprised of one partial Left open reading frame (ORF) (349 amino acids, aa) and complete Mid (578 aa) and Right (820 aa) ORFs. The amino acid sequences of the 3 ORFs were compared among isolates. The Right ORF was found to have the highest variation with a mean distance of 24%. This was followed by the Left and Mid ORF with distances of 13 and 7%, respectively. A phylogenetic analysis based on the amino acid sequence of the Right ORF divides 7 HPV isolates into 3 well-separated groups: Korea, Thailand, and Australia. The Madagascar HPV clustered with the Korea and Tanzania isolates. In Madagascar, HPV has been detected by histological examination since the 1990s. PCR analysis of a recent (2007) sampling showed a 100% prevalence. HPV was also detected in Mozambique with a 100% prevalence. High (95%) prevalence of HPV was found in wild Penaeus merguinesis collected from New Caledonia. These results indicate that HPV displays a high degree of genetic diversity and is distributed worldwide among populations of penaeid shrimp.

A quick fuse and the emergence of Taura syndrome virus

Over the last two decades, Taura syndrome virus (TSV) has emerged as a major pathogen in penaeid shrimp aquaculture and has caused substantial economic loss. The disease was first discovered in Ecuador in 1991, and the virus is now globally distributed with the greatest concentration of infections in the Americas and Southeast Asia. To determine the evolutionary history of this virus, we constructed a phylogeny containing 83 TSV isolates from 16 countries sampled over a 16-year period. This phylogeny was inferred using a relaxed molecular clock in a Bayesian Markov chain Monte Carlo framework. We found phylogenetic evidence that the TSV epidemic did indeed originate in the Americas sometime around 1991 (1988-1993). We estimated the TSV nucleotide substitution rate at 2.37 x 10(-3) (1.98 x 10(-3) to 2.82 x 10(-3)) substitutions/site/year within capsid gene 2. In addition, the phylogeny was able to independently corroborate many of the suspected routes of TSV transmission around the world. Finally, we asked whether TSV emergence in new geographic locations operates under a quick fuse (i.e. rapid appearance of widespread disease). Using a relaxed molecular clock, we determined that TSV is almost always discovered within a year of entering a new region. This suggests that current monitoring programs are effective at detecting novel TSV outbreaks.