Search

Acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease of marine shrimp that has caused severe economic losses to aquaculture producers in affected areas. AHPND was originally found to be caused by virulent strains of Vibrio parahaemolyticus (VpAHPND). These pathogenic strains carry pirABvp genes, which produce a binary toxin and are located on pVA1-type plasmid. Subsequently, it was found that AHPND can also be caused by pathogenic strains of other Vibrio species including: V. harveyi-like, V. campbellii, V. punensis, and V. owensii, all of which harbor a pVA1-type plasmid. Here we hypothesize that the pVA1-type plasmid can be transferred horizontally among Vibrio species. We carried out laboratory studies confirming the horizontal transfer of pVA1-type plasmid from AHPND-causing V. campbellii strain 20130629003S01 (VcAHPND-3S01) to a non-AHPND V. owensii strain 20160513VC2W (Vo-VC2W). ANI analysis revealed that strain Vo-VC2W is closely related to the V. owensii strain XSBZ03 isolated from corals in the South China Sea. However, the pirABvp genes on the transferred plasmid were unstable and lost from the pVA1-type plasmid of Vo-VC2W after 2 sub-culture passages. The sequences of the pVA1-type plasmid in AHPND-Vc3S01 and ΔpirABvpVo-VC2W were highly homologous (99.9% identical), indicating that this plasmid acquired by the V. owensii was from the AHPND-V. campbellii strain. In addition, complete genome sequence of the ΔpirABvpVo-VC2W showed that it contains 2 chromosomes and 3 plasmids; its detailed genomic characteristics will provide relevant information for better understanding of a V. owensii isolate that harbors transferred pVA1-type plasmid.

Tubulins are major components of microtubules, structures that are involved in many cellular processes. The gene β-tubulin, which encodes a β-tubulin protein, is conserved, and yet with sufficient variability to be useful for phylogenetic studies and for use in molecular diagnostic protocols. We sequenced and analyzed a fragment (870-bp) of β-tubulin gene of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp and associated with growth retardation. The sequence was then used to conduct a phylogenetic study to determine the position of EHP within the Microsporidia and to develop primers for an EHP nested PCR method. Blast analysis revealed a 75% nucleotide identity of EHP β-tubulin to that of E. bieneusi. Similar to other studies based on the small subunit rRNA sequence, the phylogenetic analysis using β-tubulin sequences clustered EHP with E. bieneusi and two other crustacean microsporidia, Agmasoma penaeii and Hepatospora eriocheir. As for the diagnostic use, a nested PCR method was developed that successfully detected EHP in infected Penaeus vannamei from Indonesia, Thailand, Vietnam, and India and in some cases of Artemia biomass. There were no cross-reactions to the shrimp and other crustacean genomes. This nested PCR is 100-fold more sensitive than the one-step PCR and thus suitable for detecting a low level of EHP infection.

The impacts of mariculture on coastal ecosystems are increasingly raising attention recently. To study the prevalence of white spot syndrome virus (WSSV) in wild crustaceans, a pathogenic agent of cultured shrimps, continuous and large-scale surveys in the Bohai Sea were firstly conducted from 2016 to 2018. A total of 820 samples were collected from 59 sampling sites. The results of loop-mediated isothermal amplification (LAMP) assay showed that 11 of the 19 wild species were identified to be WSSV positive. Among them, Alpheus distinguendus had the highest WSSV-positive rate at 21.9%. The percentage of WSSV positive sampling sites was 76.7%, 55.0%, 43.7% in 2016, 2017, 2018, respectively. The percentage of WSSV positive samples was 17.4%, 12.2% and 7.8%, respectively. The multiple sequence alignment and phylogenetic tree analysis showed that the WSSV VP26 gene cloned from the wild crustaceans was consistent with those from WSSV strains previously reported in India, China, Brazil, Thailand, as well as Mexico, which was clustered tightly into one branch. The presence of WSSV virions in the epithelial cells of wild southern rough shrimp Trachypenaeus curvirostris was confirmed by TEM assay, which further verifying the occurrence of WSSV infection in wild crustaceans. The results of present study demonstrated that WSSV had colonized in the populations of offshore wild crustaceans in the Bohai Sea, and the potential negative impact of WSSV prevalence on the wild crustacean populations together with marine ecosystem deserved close attention and further investigation.

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intracellular parasite that has become a critical threat to the shrimp farming industry in SE Asia. This parasite replicates in the hepatopancreas and midgut, and infected shrimp exhibit reduced feeding and severely retarded growth. In this study, we describe the first case of EHP-infected Penaeus vannamei cultured in Venezuela. Its histopathology is very similar to that of SE Asia EHP, with infected shrimp showing basophilic inclusions in hepatopancreas. Upon in situ hybridization of an EHP 18S rRNA gene fragment labeled with digoxigenin, the probe reacted intensely to the basophilic inclusions within the cytoplasm of the infected cells. We also compared the nucleotide sequence similarities in 18S rRNA gene (1095-bp), in β-tubulin (583-bp) and spore wall protein (431-bp) genes between the Venezuela EHP and SE Asia isolates; and the results showed 99%, 93% and 91% identities, respectively. This suggests that the Venezuela EHP was not recently introduced from SE Asia. In a shrimp farm that was affected by EHP, Taura syndrome virus (TSV) was also detected in the P. vannamei by RT-qPCR and histological examinations. TSV is a major pathogen in penaeid shrimp aquaculture and has caused substantial economic losses during the last 2 decades, however, TSV has not been reported since 2011. This TSV was determined to be a Venezuela genotype based on the phylogenetic analysis in the capsid protein 2 gene and it showed to have sequence identities of 97% (nucleotide) and 98% (amino acid) to a Venezuela isolate found in 2005. The emergence of EHP and occurrence of TSV in the Venezuela will have a significant impact on the shrimp production if they spread to other farms. Therefore, the shrimp cultured within the country should be monitored closely for the presence of EHP and TSV.

The hepatopancreatic microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that affects cultured shrimp Penaeus vannamei in several SE Asian countries including China, Vietnam, Thailand, Indonesia, India and Malaysia. EHP infections are often accompanied by opportunistic infections of Vibrio spp. Laboratory challenges and a case control study were used to determine the effects of EHP infection on two Vibrio diseases: acute hepatopancreatic necrosis disease (AHPND) and septic hepatopancreatic necrosis (SHPN). To determine the effect of EHP on AHPND, two independent experimental infections were carried out. EHP-infected shrimp (EHP-AHPND group) and healthy shrimp (AHPND group) were challenged with 2.4 × 105 CFU/mL of AHPND-causing Vibrio parahaemolyticus. The results of the experimental infections showed that EHP-AHPND group exhibited higher mortalities (60 and 44%) than the AHPND group (0 and 18%). The pathological effects of AHPND vs. EHP-AHPND groups were compared during the first 12 h post infection, 57% of the EHP-AHPND group displayed severe hepatopancreas necrosis and sloughing, features characteristic of AHPND infection while only 11% of AHPND group showed these features. This indicated that EHP-infected shrimp have a higher susceptibility to AHPND infection. To determine the effect of EHP on SHPN, we reviewed the histopathology of samples collected where EHP is endemic; and a case control study was carried out to determine the association between SHPN and EHP. We compared individual shrimp displaying histological signs of SHPN with the shrimp from the same ponds without these signs. A strong association was found between SHPN and EHP, indicating that shrimp with EHP have an increased susceptibility to SHPN. These findings suggested that EHP infection is a risk factor for both AHPND and SHPN.

Two Vibrio parahaemolyticus virulence genes, pir A vp and pir B vp , are known to encode a binary photorhabdus insect-related (Pir) toxin that causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. These genes are flanked with repeats of a mobile element (insertion sequence) in a large plasmid. This insertion sequence is closely (92%) related to the known insertion sequence ISVal1. The pir AB vp genes and the flanking ISVal1 forms a 5535-bp composite transposon, designated Tn6264. There are pir AB vp gene deletions in some strains of V . parahaemolyticus . During 2013–2016, we found 2 types of pir AB vp deletion mutants from AHPND-affected farms. The type I mutants included 3 strains with deletions (4.4-kb or 6.0-kb) of entire pir AB vp genes and the downstream ISVal1, and these mutants were named pir AB vp (‐). The type II mutants included 3 strains with smaller deletions (1.5-kb or 1.6-kb) including a pir A vp gene and a partial pir B vp gene, and were named pir A vp (‐). In laboratory bioassays, these were not pathogenic to shrimp confirming that both pir A vp and pir B vp are required for AHPND pathogenicity. During 2016, we also isolated 4 V . campbellii strains carrying pir AB vp genes from diseased shrimp. These V . campbellii stains were found, through laboratory bioassays and histological evaluation, to cause AHPND.

Acute hepatopancreatic necrosis disease (AHPND, also known as early mortality syndrome, EMS) caused by Vibrio parahaemolyticus has resulted in severe marine shrimp mortality and significant economic losses in related aquaculture throughout Southeast Asia and Central America. As no viable remedy has yet been reported, the main objective of this study was to develop an effective bacteriophage (phage)-based method of controlling AHPND/EMS. To determine the bacteriolytic activity of phage pVp-1 against V. parahaemolyticus strains causing AHPND/EMS, its infectivity was tested on 22 strains isolated from geographically diverse regions (5 of the Asian type and 17 of the Mexican type). This phage was able to infect 90.9% (20 strains among 22 strains) of the AHPND/EMS-related V. parahaemolyticus strains used in this study, and demonstrated substantial bacteriolytic activity against three strains known to be highly pathogenic. To the best of our knowledge, this is the first report of a virulent phage infecting V. parahaemolyticus strains responsible for AHPND/EMS, and indicates the potential utility of pVp-1 in phage therapy.

A quantitative polymerase chain reaction (qPCR) assay was developed, based on a TaqMan probe, to detect and quantify a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can cause acute hepatopancreatic necrosis disease (AHPND). The assay uses a pair of PCR primers, which amplify a 135-bp DNA fragment, and a TaqMan probe selected from the plasmid pir A-like gene. This qPCR assay reacted with AHPND-pathogenic isolates of V. parahaemolyticus collected from Vietnam and Mexico, but not with non-pathogenic strains of Vibrio spp. For quantification, a plasmid (pVpPirA-1) containing the target pir A-like gene was constructed, purified and serially diluted to be used as a standard. With this standard, the qPCR assay was then used to quantify the virulence plasmid in shrimp samples collected from different farms. Up to 5.8 × 10 5 copy per mg tissue were detected in AHPND-affected shrimp collected from Vietnam. Lower quantities, up to 1.5 × 10 4 copies per mg of tissues were detected in affected shrimp collected from a Chinese farm. In the laboratory bioassays, similar plasmid quantities, 1.8 × 10 3 to 4.7 × 10 6 copies of plasmid per mg of tissues were found in the moribund/dead shrimp, 3.5 × 10 2 to 2.2 × 10 6 copies of plasmid per mL were detected in the water samples. This assay is specific with high sensitivity (10 copies of virulence plasmid) and can be used to detect AHPND-pathogenic V. parahaemolyticus in shrimp and water samples.

A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in Penaeus vannamei . A pair of primers which amplify a 67 bp DNA fragment and a TaqMan probe were selected from the 16 S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 10 8 copies. An NHP-B challenge test using two different populations, Colombia (COL) and Specific Pathogen Free KONA line (KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs 1 × 10 5 copies).

In early 2005, the Aquaculture Pathology Laboratory of the University of Arizona received samples of diseased shrimp, Penaeus vannamei, from a Taura syndrome (TS) outbreak in the Lake Maracaibo region of Venezuela. Histopathology and in situ hybridization (ISH) were performed and the results confirmed the presence of Taura syndrome virus (TSV). The viral isolate was sequenced and presented a 93% similarity with the TSV reference strain from Hawaii (TSV-HI94). Immunohistochemistry (IHC), dot blot immunoassay and bioassays were also performed. While processed samples reacted only faintly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassay, the Venezuelan isolate of TSV (TSV-VE05) presented mortality comparable to TSV-HI94 in P. vannamei SPF Kona stock. These data indicate that a new variant of the virus was responsible for the outbreak of TS in Venezuela.

Infectious myonecrosis, caused by Infectious myonecrosis virus (IMNV), is an important emerging disease of shrimp that has affected the production of cultured Litopenaeus vannamei in Northeast Brazil. In this study we report the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe to detect this virus in shrimp. The real-time RT-PCR showed a strong linear correlation (r2 = 0.986) between threshold cycles (CT) and RNA quantities. The assay gave negative results for other viruses, including Yellow head virus (YHV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), and White spot syndrome virus (WSSV) and the necrotizing hepatopancreatic bacterium (NHPB). This real-time RT-PCR assay can detect as few as 10 IMNV copy numbers/μl RNA, while the nested RT-PCR can detect no fewer than 1000 IMNV copy numbers/μl RNA. Specific-pathogen-free L. vannamei were used in the infectivity assay. There were one control group (Group 1) and one viral challenged group (Group 2), from which shrimp were sampled for RT-PCR and histological analysis. The RNA from dead shrimp was extracted and tested for IMNV by nested and real-time RT-PCR. The shrimp in Group 1 showed 100% survival, while those in Group 2 showed a 0% survival. The first mortality in the Group 2 was observed at Day 8 post-inoculation (p.i.); and the mortalities dramatically increased after Day 40 p.i. Histological sections from Group 2 shrimp taken at intervals throughout the study exhibited acute to chronic phase lesions of IMNV infection, and consecutive tissue sections reacted to the IMNV-specific cDNA probes by in situ hybridization. The real-time RT-PCR detected the presence of IMNV in all 30 of the challenged specimens in Group 2. In contrast, the nested RT-PCR detected the presence of IMNV in 23 of the 30 specimens. The real-time RT-PCR revealed that the 7 specimens not detected by nested RT-PCR contained relatively low IMNV copy numbers compared to the other 23 specimens. These results demonstrate that the real-time RT-PCR developed in this study is a sensitive diagnostic method for IMNV.

Penaeus monodon-type baculovirus (MBV) infects and causes disease in hatchery-reared larvae and early stages of juvenile P. monodon. In this study, an improved 1-step PCR method was developed for the detection of MBV. The oligonucleotide primers, assigned as 261F and 261R, were designed from a genomic clone of MBV. Using extracted DNA from hepatopancreas or feces of ten geographic isolates of MBV infected shrimp, the PCR reaction yields a 261 bp fragment specific to the MBV sequence. Primers 261F/R did not cross-react with DNA from other shrimp viruses or with uninfected shrimp tissue. The detection limit of this primer pair is 100 copies of positive control plasmid DNA. This improved molecular method provides a diagnostic tool for identification of MBV in infected shrimp tissue collected from different geographic regions.

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.

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.

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.

Taura syndrome virus (TSV)-specific primers, designated as 7171F/7511R, were designed to improve the sensitivity of RT-PCR detection. This pair of primers was shown to detect TSV isolates representative of four phylogenetic lineages: Belize, Americas, SE Asia, and Venezuela. Its detection limit was determined to be 20 copies of the TSV genome, 100 times more sensitive than the TSV primers 9992/9195 currently being used by many laboratories and recommended for surveillance and diagnostic applications by the Office International des Epizooties. Primers 7171F/7511R were found to be specific to TSV and did not to react to either Infectious hypodermal and hematopoietic necrosis virus (IHHNV), White spot syndrome virus (WSSV), Yellow head virus (YHV), or Infectious myonecrosis virus (IMNV). This new RT-PCR method was shown to detect TSV in chronically infected Penaeus vannamei that had survived up to 236 days after exposure to TSV.