Carlos R. Pantoja

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.

A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Kochs Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.

Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico.

Acute hepatopancreatic necrosis disease (AHPND), which has also been referred to as early mortality syndrome (EMS), initially emerged as a destructive disease of cultured shrimp species in Asia in 2009. The pathogen associated with the disease, Vibrio parahaemolyticus, subsequently spread to the Western Hemisphere and emerged in Mexico in early 2013. The spread to the Western Hemisphere is a major concern to shrimp producers in the region. To date, the only peer-reviewed published method for determining whether mortalities are due to AHPND is through histological examination. A novel PCR detection method was employed to assess samples from Mexico in order to confirm the presence of the pathogen in this country. This manuscript details the detection methods used to confirm the presence of AHPND in Mexico. Both immersion and per os challenge studies were used to expose the Penaeus vannamei to the bacteria in order to induce the disease. Histological analysis confirmed AHPND status following the challenge studies. Also provided are the details of the molecular test by PCR that was used for screening candidate V. parahaemolyticus isolates. A rapid PCR assay for detection of AHPND may help with early detection and help prevent the spread of AHPND to other countries.

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.

Similarity between the histopathology of white spot syndrome virus and yellow head syndrome virus and its relevance to diagnosis of YHV disease in the Americas

The similarity of lesions, especially of the lymphoid organ (LO), caused by severe white spot syndrome virus (WSSV) infection and those once thought to be diagnostic for yellow head virus (YHV) infection have been the cause of misdiagnosis of YHV disease at several pathology laboratories in the Americas. In order to illustrate this similarity, and to demonstrate the risk of misdiagnosis, specimens of shrimp Penaeus setiferus and P. vannamei, experimentally infected with WSSV, were examined by conventional H&E histology and by in situ hybridization (ISH) with nonradioactively labeled gene probes specific for detection of each virus. H&E analysis of shrimp with severe WSSV infection revealed the presence of severe necrosis of the lymphoid organ and of the fibrous connective tissue that were very similar to that caused by YHV infection. Five representative shrimps, displaying severe lymphoid organ necrosis, were selected and subjected to in situ hybridization with the gene probe specific for detection of YHV, and the results were negative. When the same specimens were subjected to in situ hybridization with the WSSV-specific gene probes, a strong positive reaction to the probes was observed both systemically and within the lymphoid organ. Clinical samples of 10 shrimps originating from several Latin American countries and diagnosed with severe WSSV infection by H&E histology and displaying severe lymphoid organ necrosis were also examined by in situ hybridization. No reaction to the YHV probe was observed in any of these specimens. Hence, we have concluded that severe infection by WSSV may result in systemic necrosis, and necrosis of the lymphoid organ, in particular, which is very similar to that caused by YHV. Since the presence of YHV has not been confirmed in the Americas, it is recommended that in cases when YHV infection may be suspected after conventional H&E analysis, additional evidence should be obtained by other means such as in situ hybridization with a YHV-specific probe and/or by RT-PCR.

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.

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.

Improvement of a PCR method for the detection of necrotizing hepatopancreatitis in shrimp

Necrotizing hepatopancreatitis (NHP) is considered to be one of the most important bacterial diseases affecting penaeid shrimp culture and is caused by an unclassified Gram-negative, pleomorphic, intracellular Alphaproteobacterium. Due to the enteric nature of the bacteria, PCR is the one non-lethal method available for detection of the pathogen. Over a decade ago, a PCR protocol was developed for detection of NHP, which over the subsequent years was shown to occasionally generate false positive reactions. The University of Arizona Aquaculture Pathology Laboratory has developed a set of primers and PCR cycling parameters that have been tested on a variety of DNA templates, using 2 types of PCR reagent systems, which eliminated the generation of false positive amplicons.

New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia

White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly pathogenic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Arabia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV isolates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analysis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a specific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing complete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock.

Nonsusceptibility of primate cells to Taura syndrome virus.

Primate cells commonly used to test for viruses of the Picornaviridae family are not susceptible to infection by Taura syndrome virus of penaeid shrimp.

“Candidatus Hepatobacter penaei,” an Intracellular Pathogenic Enteric Bacterium in the Hepatopancreas of the Marine Shrimp Penaeus vannamei (Crustacea: Decapoda)

ABSTRACT The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name “Candidatus Hepatobacter penaei” is proposed for these pathogenic bacteria, which are members of the Rickettsiales order.