Arun K Dhar

Interests

Teaching

Aquaculture, Diseases of Aquatic Animals, Animal model for biomedical reearch.

Research

Discovering pathogens, developing diagnostic tools and vaccines and therapies against infectious diseases in crustaceans and finfish.

Courses

Scholarly Contributions

Books

• Dhar, A. K., Cruz Flores, R., & Bateman, K. S. (2022). Viral Diseases of Crustaceans. In: Invertebrate Pathology. Andrew F. Rowley, Christopher J. Coates, Miranda M. A. Whitten. Oxford, UK: Oxford University Press.

Chapters

• Dhar, A. K. (2017). Infectious Pancreatic Necrosis Virus. In Fish Viruses AND Bacteria: Pathobiology and Protection(pp 1 to 12). CAB International.

Journals/Publications

• Alenton, R. R., Mai, H. N., & Dhar, A. K. (2023). Engineering a replication-incompetent viral vector for the delivery of therapeutic RNA in crustaceans. PNAS nexus, 2(9), pgad278.
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  Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV carrying the GFP RNA cargo resulting in MrNV-GFP. Upon incorporating MrNV-GFP in the diet of the marine Pacific white shrimp (), MrNV particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within 8 days postadministration of the viral vector, which indicated a lack of MrNV-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.
• Dhar, A. K., Cruz-Flores, R., Mai, H. N., & Warg, J. (2024). Comparison of Polymerase Chain Reaction (PCR) assay performance in detecting Decapod penstylhamaparvovirus 1 in penaeid shrimp. Journal of virological methods, 323, 114840.
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  Decapod Penstylhamaparvovirus 1, commonly known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), remains an economically important viral pathogen for penaeid shrimp aquaculture due to its effects on growth performance. The World Organization for Animal Health (WOAH, Paris, France) recommended methods for the detection of IHHNV include both conventional and real-time PCR. However, published reports and anecdotal evidence suggest the occurrence of non-specific amplifications when testing for IHHNV using the WOAH protocols. Studies were designed to develop a sensitive, robust TaqMan PCR method for detection of IHHNV in the three commercially important penaeid shrimp: Penaeus vannamei, P. monodon and P. stylirostris. We compared the performance of the WOAH-recommended real-time PCR method to several published as well as in-house designed primer/probe sets spanning the entire genome of IHHNV. Our results show that (1) more than one primer/ probe set is needed when testing for the infectious form of IHHNV in all three species of shrimp and (2) primer pairs qIH-Fw/qIH-Rv and 3144F/ 3232R have diagnostic characteristics that would enable IHHNV detection in all three shrimp species. These findings are valuable for a large-scale screening of shrimp using a TaqMan real-time PCR assay.
• Dhar, A. K., Cruz-Flores, R., Mai, H. N., Aranguren Caro, L. F., Intriago, P., & Romero, X. (2023). Detection of a novel microsporidium with intranuclear localization in farmed Penaeus vannamei from Latin America. Journal of invertebrate pathology, 200, 107968.
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  Microsporidia are emerging intracellular parasites of most known animal phyla in all ecological niches. In shrimp aquaculture, the microsporidium Enterocytozoon hepatopenaei (EHP) is a major cause of concern inflicting tremendous losses to shrimp producers in southeast Asia. During a histopathological examination of Penaeus vannamei samples originating in a country from Latin America presenting slow growth, we observed abnormal nuclei in the epithelial cells of the hepatopancreas. A PCR screening of the samples using DNA isolated from paraffin embedded tissues for the SSU rRNA gene of EHP provided a 149 bp amplicon. In situ hybridization using the SSU rRNA gene probe provided a positive signal in the nuclei instead of the cytoplasm. Sequence analysis of the SSU rRNA gene product revealed a 91.3 %, 89.2 % and 85.4 % sequence identity to Enterocytozoon bieneusi, E. hepatopenaei and Enterospora canceri respectively. Furthermore, phylogenetic analysis revealed the newly discovered microsporidium clustered with E. bieneusi. Considering the intranuclear location of the novel microsporidium and the differences in the sequence of the SSU rRNA, we tentatively consider this parasite a new member of the genus Enterospora sp. The pathogenicity and distribution of the shrimp Enterospora sp. are currently unknown. Our future efforts are focused on the characterization and development of diagnostic tools for this parasite to understand if it acts as an emergent pathogen that might require surveillance to prevent its spread.
• Lee, Y., Vijayan, J., Roh, H., Park, J., Lee, J., Luan, N., Kim, H., Kim, W., Dhar, A. K., Park, C., & Kim, D. (2023). Nucleic acid-based methods for diagnosis of shrimp viral diseases. . Review in Aquaculture, 1–31. doi:10.1111/raq.12873 
• Major, S. R., Harke, M. J., Cruz-Flores, R., Dhar, A. K., Bodnar, A. G., & Wanamaker, S. A. (2023). Rapid Detection of DNA and RNA Shrimp Viruses Using CRISPR-Based Diagnostics. Applied and environmental microbiology, 89(6), e0215122.
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  Timely detection of persistent and emerging pathogens is critical to controlling disease spread, particularly in high-density populations with increased contact between individuals and limited-to-no ability to quarantine. Standard molecular diagnostic tests for surveying pathogenic microbes have provided the sensitivity needed for early detection, but lag in time-to-result leading to delayed action. On-site diagnostics alleviate this lag, but current technologies are less sensitive and adaptable than lab-based molecular methods. Towards the development of improved on-site diagnostics, we demonstrated the adaptability of a loop-mediated isothermal amplification-CRISPR coupled technology for detecting DNA and RNA viruses that have greatly impacted shrimp populations worldwide; White Spot Syndrome Virus and Taura Syndrome Virus. Both CRISPR-based fluorescent assays we developed showed similar sensitivity and accuracy for viral detection and load quantification to real-time PCR. Additionally, both assays specifically targeted their respective virus with no false positives detected in animals infected with other common pathogens or in certified specific pathogen-free animals. The Pacific white shrimp (Penaeus vannamei) is one of the most valuable aquaculture species in the world but has suffered major economic losses from outbreaks of White Spot Syndrome Virus and Taura Syndrome Virus. Rapid detection of these viruses can improve aquaculture practices by enabling more timely action to be taken to combat disease outbreaks. Highly sensitive, specific, and robust CRISPR-based diagnostic assays such as those developed here have the potential to revolutionize disease management in agriculture and aquaculture helping to promote global food security.
• Andrade, T. P., Cruz Flores, R., Mai, H. N., & Dhar, A. K. (2022). Novel Infectious Myonecrosis Virus (IMNV) variant is associated with recent disease outbreaks in Penaeus vannamei shrimp in Brazil. 
  . Aquaculture, 554, 738159. doi:https://doi.org/10.1016/j.aquaculture.2022.738159
• Aranguren Caro, L. F., Gomez-Sanchez, M. M., Piedrahita, Y., Mai, H. N., Cruz-Flores, R., Alenton, R. R., & Dhar, A. K. (2022). Current status of infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) in the Peruvian and Ecuadorian shrimp industry. PloS one, 17(8), e0272456.
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  Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3-100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.
• Cruz-Flores, R., Andrade, T. P., Mai, H. N., Alenton, R. R., & Dhar, A. K. (2022). Identification of a Novel Solinvivirus with Nuclear Localization Associated with Mass Mortalities in Cultured Whiteleg Shrimp (). Viruses, 14(10).
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  The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family . The novel virus has been tentatively termed solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other ( virus 1) solinvivirus. A unique feature of this virus that differs from other members of the is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family .
• Cruz-Flores, R., Hernández Rodríguez, M., Flores, J. S., & Dhar, A. K. (2022). Formalin-fixed paraffin-embedded tissues for microbiome analysis in rainbow trout (Oncorhynchus mykiss). Journal of microbiological methods, 192, 106389.
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  The gut microbiomes of rainbow trout (Oncorhynchus mykiss) reared at 16° and 22 °C were determined using formalin-fixed paraffin-embedded tissues (FFPE) and compared to fresh frozen tissue. The data revealed microbiomes could be successfully determined using FFPE tissue opening a new horizon in studying intestinal microbiota using archived histological samples.
• Cruz-Flores, R., López-Carvallo, J. A., Cáceres-Martínez, J., & Dhar, A. K. (2022). Microbiome analysis from formalin-fixed paraffin-embedded tissues: Current challenges and future perspectives. Journal of microbiological methods, 196, 106476.
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  Formalin-fixed paraffin-embedded (FFPE) tissues stored in thousands of human and animal pathology laboratories around the globe represent mines of stored genetic information. In recent years, the use of FFPE tissues as a viable source of DNA for diverse genetic studies has attracted attention for interrogating microbiomes from this sample type. These studies have proven that 16S rRNA amplicon sequencing-based microbiome studies are possible from FFPE samples but present some particular challenges. In this review, we summarize all aspects of microbiome studies from FFPE tissues including the challenges associated with working highly degraded DNA, best practices for reducing environmental contamination, and we propose solutions to address these issues. Finally, we discuss how the combination of FFPE microbiome studies and Laser Capture Microdissection and/or Laser Microdissection could enable to determine the spatial heterogeneity underlying complex bacterial communities.
• Dhar, A. K., Cruz-Flores, R., Warg, J., Killian, M. L., Orry, A., Ramos, J., Garfias, M., & Lyons, G. (2022). Genetic Relatedness of Infectious Hypodermal and Hematopoietic Necrosis Virus Isolates, United States, 2019. Emerging infectious diseases, 28(2), 373-381.
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  Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a nonenveloped, linear, single-stranded DNA virus belonging to the family Parvoviridae and is a World Organisation for Animal Health (OIE)-notifiable crustacean pathogen. During screening of Penaeus vannamei shrimp from 3 commercial shrimp facilities in the United States for a panel of OIE-listed (n = 7) and nonlisted (n = 2) crustacean diseases, shrimp from these facilities tested positive for IHHNV. Nucleotide sequences of PCR amplicons showed 99%-100% similarity to IHHNV isolates from Latin America and Asia. The whole genome of the isolates also showed high similarity to type 2 infectious forms of IHHNV. Phylogenetic analysis using capsid gene and whole-genome sequences demonstrated that the isolates clustered with an IHHNV isolate from Ecuador. The detection of an OIE-listed crustacean pathogen in the United States highlights the need for biosecurity protocols in hatcheries and grow-out ponds to mitigate losses.
• López-Carvallo, J. A., Cruz-Flores, R., & Dhar, A. K. (2022). The emerging pathogen Enterocytozoon hepatopenaei drives a degenerative cyclic pattern in the hepatopancreas microbiome of the shrimp (Penaeus vannamei). Scientific reports, 12(1), 14766.
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  The microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that causes substantial economic losses in shrimp (Penaeus spp.) aquaculture worldwide. To prevent diseases in shrimp, the manipulation of the gut microbiota has been suggested. However, prior knowledge of the host-microbiome is necessary. We assessed the modulation of the microbiome (bacteria/fungi) and its predicted functions over the course of disease progression in shrimp experimentally challenged with EHP for 30 days using high throughput 16S rRNA and ITS amplicon sequencing. Infection grade was assessed for the first time by quantitative digital histopathology. According to the infection intensity, three disease-stages (early/developmental/late) were registered. During the early-stage, EHP was not consistently detected, and a high diversity of potentially beneficial microorganisms related to nutrient assimilation were found. In the development-stage, most of the shrimp start to register a high infection intensity related to a decrease in beneficial microorganisms and an increase in opportunistic/pathogenic fungi. During late-stage, animals displayed different infection intensities, showed a displacement of beneficial microorganisms by opportunistic/pathogenic bacteria and fungi related to pathogen infection processes and depletion of energetic reserves. The degenerative cyclic pattern of EHP infection and its effects on beneficial microorganisms and beneficial functions of the shrimp hepatopancreas microbiome are discussed.
• Aranguren Caro, L. F., Mai, H. N., Cruz-Florez, R., Marcos, F. L., Alenton, R. R., & Dhar, A. K. (2021). Experimental reproduction of White Feces Syndrome in whiteleg shrimp, Penaeus vannamei. PloS one, 16(12), e0261289.
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  White Feces Syndrome (WFS) is an emergent disease of penaeid shrimp (Penaeus monodon and P. vannamei) that is identified by the presence of floating white fecal strings on pond water in grow-out ponds. Although the clinical manifestations of WFS are well defined, the underling etiology remains obscure. WFS has been associated with several enteric pathogens, including Enterocytozoon hepatopenaei (EHP). The association is based on studies that found areas where WFS has been reported, the prevalence and severity of EHP infection are high. In this study, we describe an experimental reproduction of WFS in P. vannamei pre-infected with EHP and challenged with a unique isolate of Vibrio parahaemolyticus isolated from the gastrointestinal tract of a shrimp displaying WFS. Upon laboratory challenge, shrimp displaying white fecal strings and white discoloration of the gastrointestinal tract were analyzed by histopathology, in-situ hybridization and quantitative PCR. Histological analysis confirmed the lesions of EHP and septic hepatopancreatic necrosis in the hepatopancreas of shrimp exposed to both pathogens. Quantitative PCR showed shrimp infected with both EHP and V. parahaemolyticus had a significantly higher load of EHP compared to shrimp infected with EHP alone. This is the first demonstration of experimental reproduction of WFS under laboratory conditions when animals are infected with EHP and V. parahaemolyticus concurrently. The data revealed a synergistic relation between EHP and V. parahaemolyticus isolate that led to the manifestation of WFS. We propose the gross signs of WFS can be used as an indicator of the presence of EHP infection in association with a particular strain of an enteric Vibrio spp. in countries where EHP is endemic.
• Cruz-Flores, R., Mai, H. N., & Dhar, A. K. (2021). Complete genome reconstruction and genetic analysis of Taura syndrome virus of shrimp from archival Davidson's-fixed paraffin embedded tissue. Virology, 553, 117-121.
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  Davidson's-fixed paraffin-embedded (DFPE) shrimp tissue are a priceless biological resource for pathogen discovery and evolutionary studies for aquaculture disease diagnostic laboratories worldwide. Nucleic acids extracted from DFPE tissues are often not adequate for most downstream molecular analysis due to fragmentation and chemical modifications. In this study, next generation sequencing (NGS) was used to reconstruct the complete genome of three geographical isolates (Belize, Venezuela and Hawaii) of a ~10 kb length RNA virus of shrimp, Taura syndrome virus (TSV), from DFPE tissues that have been archived for 15 years. Phylogenetic analyses showed that TSV isolates from Belize, Venezuela and Hawaii formed well supported clusters with homologous isolates from the corresponding regions submitted in the GenBank database. This is the first study to demonstrate the utility of archived tissue samples for identification of RNA viruses and evolutionary studies involving a viral disease in crustaceans and opens an avenue for expediting pathogen discovery.
• Mai, H. N., Aranguren Caro, L. F., Cruz-Flores, R., & Dhar, A. K. (2021). Development of a Recombinase Polymerase Amplification (RPA) assay for acute hepatopancreatic necrosis disease (AHPND) detection in Pacific white shrimp (Penaeus vannamei). Molecular and cellular probes, 57, 101710.
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  Acute hepatopancreatic necrosis disease (AHPND) is currently the most important bacterial disease of shrimp that has caused enormous losses to the shrimp industry worldwide. The causative agent of AHPND are Vibrio spp. Carrying plasmids containing the pirA and pirB genes which encode binary toxins, PirAB. Currently, AHPND is mostly diagnosed by PCR-based platforms which require the use of sophisticated laboratory instrumentation and are not suitable for a point-of-care diagnostics. Therefore, the availability of an alternative method based on isothermal amplification would be suitable for AHPND detection outside a laboratory setting and extremely useful at a pond side location. Isothermal amplification is based on the nucleic acid amplification at a single temperature and does not require the use of a thermal cycler. In this study, we developed an isothermal Recombinase Polymerase Amplification (RPA) assay for AHPND detection targeting both pirA and pirB genes, simultaneously and evaluated the specificity and sensitivity of the assay. The assay could detect AHPND without any cross-reaction with other microbial pathogens and Specific Pathogen Free (SPF) shrimp. The limit of detection of the assay was 5 copies of pirAB genes. To evaluate the reliability of the assay in detecting AHPND, DNA from Penaeus vannamei shrimp displaying acute and chronic infection were analyzed by the RPA assay and the results were compared with SYBR Green real-time PCR assay. While there was a 100% conformity between the two assay while detecting acute phase infection, RPA appeared to be more sensitive in detecting chronic phase infection. The data suggest that RPA assay described here would be a reliable method in detecting AHPND outside a standard laboratory setting.
• Mai, H. N., Caro, L. F., Cruz-Flores, R., White, B. N., & Dhar, A. K. (2021). Differentially Expressed Genes in Hepatopancreas of Acute Hepatopancreatic Necrosis Disease Tolerant and Susceptible Shrimp (. Frontiers in immunology, 12, 634152.
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  Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic sp. carrying binary toxin genes, A and B in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in . This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of .
• Millabas, J., Mai, H. N., Lin, J., Dhar, A. K., Caro, L. F., Belder, K. D., Alrehaili, Y., Alghamdi, F., Algetham, S., & Alazwari, A. (2021). The effect of salinity on enterocytozoon hepatopenaei infection in Penaeus vannamei under experimental conditions.. BMC Veterinary Research, 17(1), 65-65. doi:10.1186/s12917-021-02778-0
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  BACKGROUND: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. RESULTS: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at three different salinities (2 ppt, 15 ppt, and 30 ppt) while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt. CONCLUSION: The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt.
• Powers, Q. M., Aranguren, L. F., Fitzsimmons, K. M., McLain, J. E., & Dhar, A. K. (2021). Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND). Journal of invertebrate pathology, 186, 107554.
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  Acute hepatopancreatic necrosis disease (AHPND) is an OIE-listed enteric disease that has continued to plague the shrimp aquaculture industry since its first discovery in 2009. AHPND is one of the biggest disease threats to the shrimp aquaculture industry along with white spot disease (WSD) which has severely impacted both crayfish and shrimp aquaculture. AHPND is caused by specific marine Vibrio spp. which carry plasmid-borne binary toxins PirA and PirB. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (Vp) to discern the potential risk that AHPND may pose to the crayfish aquaculture industry. Susceptibility was investigated by challenging Cherax quadricarinatus (Australian red claw crayfish) and Penaeus vannamei (Pacific white shrimp) with Vp in a cohabitation immersion bioassay. Upon termination of the bioassay, crayfish survival was significantly higher than shrimp survival (87% vs. 33%). Hepatopancreas dissected from experimentally challenged animals were screened for the binary toxin genes pirA and pirB by real-time and duplex conventional PCR assays, and also were examined by H&E histology for the detection of characteristic AHPND pathology. Although AHPND toxin genes pirA and pirB were detected in a subset of crayfish samples, histopathology did not reveal any pathognomonic lesions that are characteristic of AHPND in any crayfish samples examined. These findings suggest that crayfish are likely resistant to AHPND.
• Quinn, P., L, A. F., Fitzsimmons, K., Mclain, J. E., & Dhar, A. K. (2021). Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND). Journal of Invertebrate Pathology.
• Swinford, J. L., Anderson, J. D., Adami, R., & Dhar, A. K. (2021). Diagnostic Molecular Investigation of White Spot Syndrome Virus Finds No Infection in Wild White Shrimp and Brown Shrimp along the Texas Gulf Coast. Journal of aquatic animal health, 33(2), 69-76.
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  White spot syndrome virus (WSSV) is a virulent disease that disrupts shrimp farm operations throughout the world. While the United States has had only limited outbreaks of WSSV within the past several decades, it is important to ensure that this disease does not infect wild penaeid shrimp populations. In Texas, there is a potential for WSSV to spread to wild penaeid populations in the Gulf of Mexico via infected imported nonnative bait shrimp, imported broodstock, or wild crustacean hosts. Due to these potential threats, the Texas Parks and Wildlife Coastal Fisheries Division monitored WSSV in wild brown shrimp Farfantepenaeus aztecus and white shrimp Litopenaeus setiferus from seven major bay systems along the Texas coast during 2019. While no positive samples were detected from the collected shrimp, a power analysis illustrated a potential for low-level WSSV prevalence within Texas shrimp populations that would not be detectable by this monitoring survey. Overall, WSSV does not appear to be a major threat in the Texas region of the Gulf of Mexico, but continual observation and monitoring of wild penaeid shrimp is necessary to protect this resource from future WSSV outbreaks.
• Agnew, M. V., Friedman, C. S., Langdon, C., Divilov, K., Schoolfield, B., Morga, B., Degremont, L., Dhar, A. K., Kirkland, P., Dumbauld, B., & Burge, C. A. (2020). Differential Mortality and High Viral Load in Naive Pacific Oyster Families Exposed to OsHV-1 Suggests Tolerance Rather than Resistance to Infection. Pathogens (Basel, Switzerland), 9(12).
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  Pacific oysters, , are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively "µvars"), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower (43% and 71%, respectively) than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 10). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.
• Aranguren Caro, L. F., Mai, H. N., Kanrar, S., Cruz-Flores, R., & Dhar, A. K. (2020). A Mutant of AB (+) That Carries Binary Toxin Genes but Does Not Cause Acute Hepatopancreatic Necrosis Disease. Microorganisms, 8(10).
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  carrying binary toxin genes, AB, is one of the etiological agents causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. This disease has emerged recently as a major threat to shrimp aquaculture worldwide. During a routine PCR screening of AHPND-causing strains, an isolate tested PCR positive for (R13) and another isolate tested positive for both the A and B (R14) genes. To evaluate the pathogenicity of these isolates, specific pathogen-free (SPF) were experimentally challenged. For both R13 and R14 isolates, the final survival rate was 100% at termination of the challenge, whereas the final survival with the AHPND-causing was 0%. The nucleotide sequence of the plasmid DNA carrying the binary toxin genes revealed that R13 contains a deletion of the entire gene whereas R14 contains the entire coding regions of both A and B genes. However, R14 possesses an insertion upstream of the A gene. In R14, mRNA for both A and B genes could be detected but no cognate proteins. This shows that the genome of AHPND-causing is highly plastic and, therefore, detection of the A and B genes alone by DNA-PCR is insufficient as a diagnostic test for AHPND.
• Aranguren Caro, L. F., Mai, H. N., Noble, B., & Dhar, A. K. (2020). Acute hepatopancreatic necrosis disease (VP), a chronic disease in shrimp (Penaeus vannamei) population raised in latin America. Journal of invertebrate pathology, 174, 107424.
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  In Latin American shrimp farming, acute hepatopancreatic necrosis disease (AHPND) does not cause the acute mortalities observed in SE Asia. Herein we report for the first time a new phase of infection of AHPND, a chronic phase based on two experimental AHPND-challenge trials using shrimp lines from Latin America. Three shrimp lines of Penaeus vannamei were challenged with a highly pathogenic strain of Vibrio parahaemolyticus causing AHPND (VP). PCR and histopathology assays were used for confirmation of AHPND in the trials. The first study was to compare survival between the lines. A follow-up trial was conducted to document hepatopancreas heterotrophic bacterial count and to measure the expression of VP binary toxin genes (pirAB genes) at 24 h.p.i. One of the Latin American shrimp lines, APE1, had significantly higher survival than recorded for the other two lines (APE2 & APE3) and the specific-pathogen-free positive control line. Histopathology showed typical AHPND acute and terminal phase lesions in VP challenged groups, although destructive cellular changes were more pronounced in the SPF line. Histopathology of animals surviving AHPND revealed a unique chronic phase of infection that resembles septic hepatopancreatic necrosis (SHPN), recognized as diagnostic of digestive tract vibriosis. Data to support our finding, including a quantitative RT-PCR assay, confirmed the expression of pirAB genes and the differential hepatopancreas heterotrophic plate count (HPC) among the different lines challenged. The results explain in part why the shrimp industry in some Latin American countries continues to grow despite the presence of AHPND. In addition, the biology and pathology of AHPND resistant/tolerant shrimp appear to be quite unique in this Latin American shrimp population.
• Aranguren Caro, L. F., Mai, H. N., Nunan, L., Lin, J., Noble, B., & Dhar, A. K. (2020). Assessment of transmission risk in WSSV-infected shrimp Litopenaeus vannamei upon cooking. Journal of fish diseases, 43(4), 403-411.
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  White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.
• Aranguren Caro, L. F., Mai, H. N., Pichardo, O., Cruz-Flores, R., Hanggono, B., & Dhar, A. K. (2020). Evidences supporting Enterocytozoon hepatopenaei association with white feces syndrome in farmed Penaeus vannamei in Venezuela and Indonesia. Diseases of aquatic organisms, 141, 71-78.
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  White feces syndrome (WFS) is an emerging and poorly described disease characterized by the presence of floating white fecal strings in shrimp (Penaeus monodon and P. vannamei) grow-out ponds. WFS has been associated with several pathogens, including Enterocytozoon hepatopenaei. This association is based on the fact that in areas where E. hepatopenaei has been reported, there was also a high WFS prevalence. E. hepatopenaei is an emerging pathogen that has affected cultured shrimp in Indonesia, Vietnam, China, Thailand, and India. In 2016, we reported the presence of E. hepatopenaei in farmed P. vannamei in Venezuela. In this study, we describe the first case of WFS in Venezuela associated with E. hepatopenaei. The white fecal strings and shrimp displaying white feces along the gastrointestinal tract observed in this study were similar to the gross signs found in WFS-impacted P. vannamei in SE Asian countries. Furthermore, we describe a strong association between WFS and E. hepatopenaei in the samples obtained from Venezuela and Indonesia. Quantification of E. hepatopenaei in WFS-affected ponds, ponds with a history of WFS, and ponds with no WFS showed that E. hepatopenaei loads were significantly higher in WFS-affected ponds. Furthermore, these findings constitute the first report of WFS being associated with E. hepatopenaei in farmed shrimp in Latin America. Additionally, we propose that the gross signs of WFS such as floating whitish fecal strings can be used as an indicator of the presence of E. hepatopenaei in countries where E. hepatopenaei is endemic.
• Burge, C. A., Reece, K. S., Dhar, A. K., Kirkland, P., Morga, B., Dégremont, L., Faury, N., Wippel, B. J., MacIntyre, A., & Friedman, C. S. (2020). First comparison of French and Australian OsHV-1 µvars by bath exposure. Diseases of aquatic organisms, 138, 137-144.
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  Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.
• Cruz-Flores, R., Mai, H. N., Kanrar, S., Aranguren Caro, L. F., & Dhar, A. K. (2020). Genome reconstruction of white spot syndrome virus (WSSV) from archival Davidson's-fixed paraffin embedded shrimp (Penaeus vannamei) tissue. Scientific reports, 10(1), 13425.
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  Formalin-fixed paraffin-embedded (FFPE) tissues are a priceless resource for diagnostic laboratories worldwide. However, DNA extracted from these tissues is often not optimal for most downstream molecular analysis due to fragmentation and chemical modification. In this study, the complete genome of white spot syndrome virus (WSSV) was reconstructed from ~ 2-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissue using Next Generation Sequencing (NGS). A histological analysis was performed on archived DFPE shrimp tissue and a sample showing a high level of WSSV infection was selected for molecular analysis. The viral infection was further confirmed by molecular methods. DNA isolated from DFPE and fresh frozen (FF) tissues were sequenced by NGS. The complete genome reconstruction of WSSV (~ 305 kbp) was achieved from both DFPE and FF tissue. Single nucleotide polymorphisms, insertion and deletions were compared between the genomes. Thirty-eight mutations were identified in the WSSV genomes from the DFPE and FF that differed from the reference genome. This is the first study that has successfully sequenced the complete genome of a virus of over 300 kbp from archival DFPE tissue. These findings demonstrate that DFPE shrimp tissue represents an invaluable resource for prospective and retrospective studies, evolutionary studies and opens avenues for pathogen discovery.
• Dhar, A. K. (2020). A cross-sectional study of shrimp pathogens in wild shrimp, Penaeus vannamei and Penaeus stylirostris in Tumbes, Peru. Aquaculture Research. doi:10.1111/are.14969
• Dhar, A. K. (2020). Impact of Acute Hepatopancreatic Necrosis Disease (AHPND) on the freshwater prawn (Macrobrachium rosenbergii) and Pacific white shrimp (Penaeus vannamei) at various salinities.. Aquaculture Research. doi:10.1111/are.15001
• Friedman, C. S., Reece, K. S., Wippel, B. J., Agnew, M. V., Dégremont, L., Dhar, A. K., Kirkland, P., MacIntyre, A., Morga, B., Robison, C., & Burge, C. A. (2020). Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants. The Science of the total environment, 739, 139752.
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  The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.
• Gangnonngiw, W., Bunnontae, M., Phiwsaiya, K., Senapin, S., & Dhar, A. K. (2020). In experimental challenge with infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), MrNV alone can cause mortality in freshwater prawn (Macrobrachium rosenbergii). Virology, 540, 30-37.
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  To overcome the lack of immortal shrimp cell lines for shrimp viral research, we constructed and tested DNA infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) often found together in freshwater prawn (M. rosenbergii) exhibiting white tail disease (WTD). Full-length cDNAs of MrNV and XSV genomic RNA were individually inserted into the baculovirus pFastBacDUAL shuttle vector. Individual Sf9 (insect cell line) transfection resulted in production of RNA (RT-PCR) and capsid proteins (immunofluorescence) for both viruses. Presence of respective virions was confirmed by density gradient purification followed by RT-PCR and transmission electron microscopy. Infectivity was by tested in immersion-challenge tests with M. rosenbergii post-larvae (PL) using both semi-purified viruses, individually or combined, and confirmed by histological analysis (morphology and immunofluorescence) and quantitative RT-PCR. Mortality accompanied by WTD lesions occurred with MrNV alone or in combination with XSV but not with XSV alone, despite its replication.
• Mai, H. N., Cruz-Flores, R., & Dhar, A. K. (2020). Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirA and PirB released from Vibrio spp. Journal of microbiological methods, 176, 106002.
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  An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirA and PirB. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirA (20 mAbs) and PirB (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirA; 7B7 and 7B9 against PirB) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirA or PirB down to 0.008 ng/μl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirA and PirB toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.
• Mai, H. N., Cruz-Flores, R., Aranguren Caro, L. F., White, B. N., & Dhar, A. K. (2020). A comparative study of Enterocytozoon hepatopenaei (EHP) challenge methods in Penaeus vannamei. Journal of invertebrate pathology, 171, 107336.
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  The microsporidium Enterocytozoon hepatopenaei (EHP) is considered as an emerging pathogen threating the shrimp industry worldwide. It is an intracellular parasite that has been associated with retarded growth syndrome and white feces syndrome in shrimp. Although the impact of EHP to the shrimp industry is well known, many aspects of host-pathogen interactions are not well understood. A major limitation in the study of EHP is the lack of a reliable method to produce large quantities of inoculum rapidly and reproducibly. The present study was designed to compare different challenge methods including intramuscular injection, oral administration, co-habitation, hepatopancreas (HP) injection and reverse gavage. The results showed that the HP injection and the reverse gavage are two promising methods to infect shrimp rapidly and generate inoculum in a reproducible manner starting with a limited amount of inoculum. Therefore, the HP injection and reverse gavage were chosen for a scale-up study. Histopathology results showed that EHP proliferated in the epithelial cells of the HP in shrimp challenged via direct injection of inoculum into HP and reverse gavage treatments. In accordance with the histopathology results, the qPCR data showed that EHP loads in the challenged shrimp increased significantly with the HP injection and reverse gavage methods. Furthermore, the histopathological and quantification results indicate that HP injection and reverse gavage are two novel methods that can be used in EHP-challenge studies and for rapidly generating viable EHP inoculum.
• Ochoa, L. M., Cruz-Flores, R., & Dhar, A. K. (2020). Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson's-Fixed Paraffin-Embedded Shrimp Tissue. Viruses, 12(9).
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  Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection and phylogenetic analyses. Total RNA was isolated from known TSV-infected DFPE tissues using three commercially available kits and the purity and ability to detect TSV in the isolated RNA were compared. TSV was successfully detected through RT-qPCR in all the tested samples. Among the TSV-specific primers screened through RT-PCR, primer pair TSV-20 for the RNA-dependent RNA polymerase (RdRp), primers TSV-15 and TSV-16 for the capsid protein gene VP2 and primers TSV-5 for the capsid protein gene VP1 amplified the highest number of samples. To assess the phylogenetic relation among different TSV isolates, the VP1 gene was amplified and sequenced in overlapping segments. Concatenated sequences from smaller fragments were taken for phylogenetic analyses. The results showed that the TSV isolates from this study generally clustered with homologous isolates from the corresponding geographical regions indicating RNA derived from DFPE tissues can be used for pathogen detection and retrospective analyses. The ability to perform genomic characterization from archived tissue will expedite pathogen discovery, development of diagnostic tools and prevent disease spread in shrimp and potentially other aquaculture species worldwide.
• Orchard, J. D., Cetlin, D., Pallansch, M., Barlow, R., Borman, J., Dhar, A., Pallansch, L., & Dickson, M. (2020). Using a noninfectious MVM surrogate for assessing viral clearance during downstream process development. Biotechnology progress, 36(1), e2921.
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  Viral contamination is an inherent risk during the manufacture of biopharmaceuticals. As such, biopharmaceutical companies must demonstrate the viral clearance efficacy of their downstream process steps prior to clinical trials and commercial approval. This is accomplished through expensive and logistically challenging spiking studies, which utilize live mammalian viruses. These hurdles deter companies from analyzing viral clearance during process development and characterization. We utilized a noninfectious minute virus of mice-mock virus particle (MVM-MVP) as a surrogate spiking agent during small scale viral filtration (VF) and anion exchange chromatography (AEX) studies. For VF experiments, in-process mAb material was spiked and processed through Asahi Kasei P15, P20, P35, and BioEX nanofilters. Across each filter type, flux decay profiles and log reduction values (LRVs) were nearly identical for either particle. For AEX experiments, loads were conditioned with various amounts of sodium chloride (9, 20, 23, and 41 mS/cm), spiked with either particle and processed through a Q-SFF packed column. LRV results met our expectations of predicting MVM removal.
• Cruz-Flores, R., Mai, H. N., & Dhar, A. K. (2019). Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB. Molecular and cellular probes, 43, 20-28.
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  Acute hepatopancreatic necrosis disease (AHPND), also known as Early mortality syndrome (EMS), is a recently emerged lethal disease that has caused major economic losses in shrimp aquaculture. The etiologic agents are Vibrio spp. that carry Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB. A multiplex SYBR Green real-time PCR was developed that detects pirA, pirB, and two internal control genes, the shrimp 18S rRNA and the bacterial 16S rRNA genes in a single reaction. The pirB primers amplify the 3'-end of the pirB gene allowing the detection of Vibrio spp. mutants that contain a complete deletion of pirA and the partial deletion of pirB. The assay also detects mutants that contain the entire pirA gene and the deletion of the pirB gene. Since both toxin genes are needed for disease development, this assays can distinguish between pathogenic strains of Vibrio spp. that cause AHPND in shrimp and mutants that do not cause disease. The amplicons for pirA, pirB, 18S rRNA and 16S rRNA showed easily distinguishable melting temperatures of 78.21 ± 0.18, 75.20 ± 0.20, 82.28 ± 0.34 and 85.41 ± 0.21 °C respectively. Additionally, a duplex real-time PCR assay was carried out by designing TaqMan probes for the pirA and pirB primers. The diagnostic sensitivity and specificity was compared between the SYBR Green and TaqMan assays. Both assays showed similar sensitivity with a limit of detection being 10 copies for pirA and pirB, and neither assays showed any cross reaction with other known bacterial and viral pathogens in shrimp. The high sensitivity of both assays make them suitable for the detection of low copies of the pirA and pirB genes in AHPND causing Vibrio spp. as well as for detecting non-pathogenic mutants.
• Cruz-Flores, R., Mai, H. N., Noble, B. L., Schofield, P. J., & Dhar, A. K. (2019). Detection of Enterocytozoon hepatopenaei using an invasive but non-lethal sampling method in shrimp (Penaeus vannamei). Journal of microbiological methods, 162, 38-41.
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  The detection of enteric pathogens that cause diseases in shrimp involves the sacrifice of the host to obtain tissue samples for diagnosis. In this study, we describe an invasive but non-lethal sampling methodology using a syringe to collect biopsy samples from the hepatopancreas (HP) of Penaeus vannamei to detect the microsporidian pathogen, Enterocytozoon hepatopenaei (EHP), by qPCR and transmission electron microscopy. EHP was detected in all the infected shrimp by qPCR. The shrimp infected by the microsporidian showed 65% survival at 7 days post-sampling. Transmission electron microscopic examination of the biopsy samples revealed numerous spores of the pathogen. The presence of EHP was further confirmed by histology and in situ hybridization from HP tissue samples. The data shows that a hepatopancreas biopsy could be a viable means of detecting enteric pathogens in shrimp, and the method could be valuable in sampling broodstock and natural populations without the need to sacrifice the animals.
• Dhar, A. K., Cruz-Flores, R., Caro, L. F., Siewiora, H. M., & Jory, D. (2019). Diversity of single-stranded DNA containing viruses in shrimp. Virusdisease, 30(1), 43-57.
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  Over the past four decades, shrimp aquaculture has turned into a major industry providing jobs for millions of people worldwide especially in countries with large coastal boundaries. While the shrimp industry continues to expand, the sustainability of shrimp aquaculture has been threatened by the emergence of diseases. Diseases caused by single-stranded DNA containing viruses, such as infectious hypodermal and hematopoietic necrosis virus (IHHNV) and hepatopancreatic parvovirus (HPV), have caused immense losses in shrimp aquaculture since the early 1980s. In fact, the disease outbreak in the blue shrimp ( caused by IHHNV in early 1980s ultimately led to the captive breeding program in shrimp being shifted from to the white shrimp (), and today is the preferred cultured shrimp species globally. To date, four single-stranded DNA viruses are known to affect shrimp; these include IHHNV, HPV, spawner-isolated mortality virus (SMV) and lymphoidal parvo-like virus (LPV). Due to the economic losses caused by IHHNV and HPV, most studies have focused on these two viruses, and only IHHNV is included in the OIE list of Crustacean Diseases. Hence this review will focus on IHHNV and HPV. IHHNV and HPV virions are icosahedral in morphology measuring 20-22 nm in size and contain a single-stranded DNA (ssDNA) of 4-6 kb in size. Both IHHNV and HPV are classified into the sub-order family . The genome architecture of both viruses are quite similar as they contain two completely (as in IHHNV) or partially overlapping (as in HPV) non-structural and one structural gene. Histopathology and polymerase chain reaction (PCR)-based methods are available for both viruses. Currently, there is no anti-viral therapy for any viral diseases in shrimp. Therefore, biosecurity and the use of genetically resistant lines remains as the corner stone in the management of viral diseases. In recent years, gene silencing using the RNA interference (RNAi) approach has been reported for both IHHNV and HPV via injection. However, the delivery of RNAi molecules via oral route remains a challenge, and the utility of RNAi-based therapy has yet to be materialized in shrimp aquaculture.
• Dhar, A. K., Piamsomboon, P., Aranguren Caro, L. F., Kanrar, S., Adami, R., & Juan, Y. S. (2019). First report of acute hepatopancreatic necrosis disease (AHPND) occurring in the USA. Diseases of aquatic organisms, 132(3), 241-247.
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  In June 2017, mass mortalities were reported at whiteleg shrimp Penaeus vannamei farms in Texas, USA. PCR testing for OIE-listed and non-listed pathogens detected the pirA and pirB toxin genes associated with acute hepatopancreatic necrosis disease (AHPND). DNA sequence analyses of cloned pirA and pirB genes showed them to be identical to those detected in other AHPND-causing Vibrio sp. Amplicons generated using PCR tests targeted to the toxR gene showed the Pir toxin genes to be associated with a V. parahaemolyticus type more similar to a genotype found in Mexico compared to that found in Asia. Histology detected masses of bacteria and hemocytic infiltrations as well as extensive necrosis and sloughing of epithelial cells in hepatopancreatic tubules pathognomonic of AHPND. The data support AHPND as the cause of the mortalities. Given that US companies produce shrimp broodstock for farms in Asia and Latin America, the further spread of AHPND in the USA needs to be prevented to avoid serious economic consequences to these industries.
• Mai, H. N., Hanggono, B., Caro, L. F., Komaruddin, U., Nur'aini, Y. L., & Dhar, A. K. (2019). Novel infectious myonecrosis virus (IMNV) genotypes associated with disease outbreaks on Penaeus vannamei shrimp farms in Indonesia. Archives of virology, 164(12), 3051-3057.
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  Infectious myonecrosis virus (IMNV) is one of the most pathogenic viruses that affect Penaeus vannamei shrimp. In 2018, IMNV was reported in grow-out ponds of P. vannamei in Situbondo, Indonesia. Diseased animals displayed clinical signs of infectious myonecrosis (IMN) characterized by white discoloration of skeletal muscle. Histopathology of affected shrimp revealed lesions that are pathognomonic of IMNV infection. The major capsid protein (MCP) gene was amplified and sequenced from representative samples showing IMN pathology. Multiple alignment of predicted amino acid sequences of the MCP gene with known IMNV genotypes in the GenBank database revealed three unique genotypes, SB-A, SB-B and SB-C,in Situbondo samples. The number of amino acid changes in SB-A, SB-B and SB-C compared to known IMNV genotypes ranged from 7-710, including the isolate SB-B, which contains deletion of 622 aa. A phylogenetic analysis using homologous sequences from Brazil and Indonesia showed that these three isolates represent new IMNV genotypes.
• Mohamed, K., Megahed, M. E., Elmesiry, G., & Dhar, A. K. (2019). Genetic parameters of survival in four generations of Indian white shrimp (Fenneropeneaus indicus). Journal of Applied Aquaculture, 31(2), 121-139. doi:10.1080/10454438.2018.1551169
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  ABSTRACTThis study evaluated the genetic variance for survival at harvest of Indian white shrimp (Fenneropenaeus indicus) using a pedigreed synthetic population generated upon collecting broodstock from different fishery grounds in Egypt. The estimated heritabilities (h2 ± SE) of survival from generations G0 to G3 ranged from 0.144 ± 0.026 to 0.008 ± 0.030 using a model that included the common environmental effect (c2). Low heritability (0.014 ± 0.015) was estimated across generations with the inclusion of common environmental effect (c2). However, the h2 of survival from G0 to G1 ranged from 0.569 ± 0.071 to 0.410 ± 0.011 when c2 was not included. The genetic correlation for survival between different ponds from G0 to G3 was moderate to high (0.79 ± 0.042 to 0.83 ± 0.012) but significantly different (P < 0.05). There were low correlation coefficients (−0.237 ± 0.089 to 0.316 ± 0.073) between body weight estimated breeding value (EBV) and survival EBV of families in four generations.
• Sullivan, T. J., Dhar, A. K., Cruz-Flores, R., & Bodnar, A. G. (2019). Rapid, CRISPR-Based, Field-Deployable Detection Of White Spot Syndrome Virus In Shrimp. Scientific reports, 9(1), 19702.
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  Rapid, sensitive, point-of-care diagnostics are critical for managing infectious diseases. Here we adapt the CRISPR-based SHERLOCK method to develop a rapid, accurate, single copy detection assay for White Spot Syndrome Virus, the most devastating virus impacting global shrimp aquaculture. Further, we combine paper matrix nucleic acid extraction and lateral flow colorimetric reporting to create a fully field-deployable, next-generation diagnostic with potential to transform veterinary pathology, disease ecology, and animal production.
• Aranguren, L. F., & Dhar, A. K. (2018). Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and quantitative PCR assays. Diseases of aquatic organisms, 131(1), 49-57.
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  Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 108 copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.
• Dhar, A. K. (2018). A comprehensive look at the Proficiency Test for farmed shrimp.. Global Aquaculture Advocate.
• Dhar, A. K. (2018). Complete Genome Sequence of a Deletion Mutant of Vibrio parahaemolyticus from Pacific White Shrimp (Penaeus vannamei).. Genome Announcement.
• Dhar, A. K. (2018). Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and real-time quantitative PCR assays.. Diseases in Aquatic Organisms.
• Dhar, A. K. (2018). Development of New PCR & Real-time PCR for Detection, Quantification of Necrotising hepatopancreatitis Bacteria (NHPB).. Global Aquaculture Advocate.
• Dhar, A. K. (2018). Genetic parameters of survival for four generations in Indian white shrimp (Fenneropeneaus indicus).. Journal of Applied Aquaculture.
• Dhar, A. K. (2018). Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics.. Journal Virological Methods.
• Dhar, A. K. (2018). The history and future of the Aquaculture Pathology Laboratory. .. Global Aquaculture Advocate.
• Dhar, A. K. (2018). Use of a noninfectious surrogate to predict minute virus of mice removal during nanofiltration.. Biotechnol Prog. 34:1213-1220..
• Kanrar, S., & Dhar, A. K. (2018). Complete Genome Sequence of a Novel Mutant Strain of Vibrio parahaemolyticus from Pacific White Shrimp (Penaeus vannamei). Genome announcements, 6(24).
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  The acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by carrying toxin genes, and We report the complete genome sequence of the novel strain R14, which did not display AHPND symptoms in despite containing the binary toxin genes.
• Weerachatyanukul, W., Senapin, S., Pudgerd, A., Jariyapong, P., Hirono, I., Dhar, A. K., & Chotwiwatthanakun, C. (2018). Construction of an infectious Macrobrachium rosenbergii nodavirus from cDNA clones in Sf9 cells and improved recovery of viral RNA with AZT treatment. Aquaculture, 483, 111-119. doi:10.1016/j.aquaculture.2017.10.008
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  Abstract Macrobrachium rosenbergii nodavirus (MrNV) is usually accompanied by extra small virus (XSV) in natural outbreaks of white tail disease (WTD) in the giant river prawn Macrobrachium rosenbergii . Testing the virulence of MrNV alone has been problematic due to the difficulty in completely separating XSV from MrNV by viral purification steps from naturally infected shrimp. However, based on reports of natural M . rosenbergii specimens from WTD outbreak ponds that were positive for MrNV but negative for XSV led us to hypothesize that MrNV alone might cause WTD. To test this hypothesis, we prepared the two, complete genomic RNA fragments (RNA1 and RNA2) of MrNV from cDNA clones and used these to transfect Sf9 cells that subsequently showed cellular changes, including cell swelling, syncytial cell formation, and development of cytoplasmic inclusions within 72 h post-transfection. Replication of RNA1 and RNA2 increased in the transfected cells and transmission electron microscopy of the cell lysates revealed the presence of icosahedral viral-like particles that were 40–50 nm in diameter. When naive Sf9 cells were inoculated with the cell lysate, the newly infected cells showed cellular changes and produced strong immunoreactivity against MrNV capsid protein indicating the infectious nature of the cell lysate. When the lysates were injected into the whiteleg shrimp Penaeus vannamei , MrNV RNA replication in the shrimp was followed by morality accompanied by typical MrNV lesions that gave possible positive immunohistochemical reactions for the MrNV capsid protein. Treatment of the Sf9 cells with azidothymidine triphosphate (AZT) prior to transfection significantly increased viral RNA synthesis and pathogenicity when compared with untreated, transfected cells. Using this model to produce infectious MrNV without XSV contamination proves that MrNV alone can be lethal to shrimp and it opens the way to further investigate the molecular basis of MrNV pathogenesis, and to develop antiviral strategy to control white tail disease.
• Dhar, A. K. (2017). Construction of an infectious Macrobrachium rosenbergii nodavirus from cDNA clones in Sf9 cells and improved recovery of viral RNA with AZT treatment.. Aquaculture, 483, 111-119.
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  Arnon Pudgerd, Wattana Weerachatyanukul, Ikuo Hirono, Saengchan Senapin, Arun K. Dhar, Charoonroj ChotwiwatthanakunConstruction of an infectious Macrobrachium rosenbergii nodavirus from cDNA clones in Sf9 cells and improved recovery of viral RNA with AZT treatmentHighlight•A complete genomic RNAs of Macrobrachium rosenbergii nodavirus was synthesized from cDNA clones by in vitro transcription.•The MrNV virions were successfully rescued from RNAs transfected insect cells.•The intact recovered MrNV virions showed their infectivity in both insect and shrimp cells.•AZT treatment drastically increased in MrNV RNAs biosynthesis.
• Johnson, S., Brorson, K. A., Frey, D. D., Dhar, A. K., & Cetlin, D. A. (2017). Characterization of Non-Infectious Virus-Like Particle Surrogates for Viral Clearance Applications. Applied biochemistry and biotechnology, 183(1), 318-331.
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  Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral "spiking" studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies.

Proceedings Publications

• Dhar, A. K., & Mai, H. N. (2021). Acute Hepatopancreatic Necrosis Disease (AHPND) and Hepatopancreatic Microsporidiosis (HPM): Two Threats to Sustainable Shrimp Aquaculture.. In Sustainable Aquaculture & Resource Enhancement, SEAFDEC, Iloilo, Philippines, June 24-26, 2019..

Presentations

• Aranguren Caro, L. F., Mai, H. N., Schofield, P., Alenton, R., & Dhar, A. K. (2021, August). Development of an Enterocytozoon hepatopenaei (EHP) challenge model in screening genetic lines of Penaeus vannamei shrimp. Aquaculture America, World Aquaculture Society Conference, San Antonio, TX, August 11-14, 2021.. San Antonio, Texas: WAS.
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  Herein, we report an E. hepatopenaei experimental challenge model using two Specific Pathogen Free (SPF) Penaeus vannamei shrimp lines originating from different geographic locations. The shrimp (weight range 6.0 to 7.0 g) from each population were tagged with a unique elastomer tag, maintained in the same tank before exposing to fecal strings from known EHP-infected shrimp as inoculum. Fecal strings were collected daily by siphoning from one 1000 L tank containing 60 EHP-infected shrimp. Histopathology and real-time PCR assays were conducted for confirmation of EHP infection in the challenged animals. The final survival was equivalent in both population (i.e. ~90%), but the prevalence of EHP varied between the two populations. SPF1 had significantly higher growth than SFF2 line (for both EHP-challenged and unchallenged treatments tanks).
• Dhar, A. K., Cruz Flores, R., & Mai, H. N. (2021, August). Expediting pathogen discovery in shrimp by combining histopathology and genomic tools.. Aquaculture America, World Aquaculture Society Conference, San Antonio, TX, August 11-14, 2021..
• Mai, H. N., Alenton, R. R., & Dhar, A. K. (2021, August). Identification of receptors of Pirabvp binary toxin released by Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) IN Penaeus vannamei shrimp.. Aquaculture America, WAS Conference, San Antonio, TX, August 11-14, 2021..
• Schofield, P., Marcos, F., Aranguren Caro, L. F., & Dhar, A. K. (2021, August). Rapid amplification of Enterocytozoon hepatopenaei (EHP) inoculum in Penaeus vannamei shrimp.. Aquaculture America, World Aquaculture Society Conference, San Antonio, TX, August 11-14, 2021..
• Dhar, A. K. (2020, November). Diseases & Disease Management in Shrimp Aquaculture. Shrimp School- 2020 (Virtual Conference). USA: National Fisheries Institute, Washington DC.
• Dhar, A. K. (2020, November). Expediting Pathogen Discovery by Combining Histopathology & Genomics. Webinar on Aquatic Animal Health. Central Institute of Fisheries Education (CIFE), Mumbai, India: Indian Council of Agricultural Research (ICAR) & Central Institute of Fisheries Education (CIFE), India.
• Dhar, A. K. (2020, September). Expediting Pathogen Discovery by Combining Conventional Pathology and Genomics. Virtual Conference on Aquaculture Techniques and Disease Management. India: Sathyabama Institute of Science & Technology University, Chennai, India.
• Cruz-Flores, R., Mai, H., Aranguren Caro, L. F., & Dhar, A. K. (2019, February). HEPATOPANCREAS TISSUE BIOPSY: AN INVASIVE BUT NON-LETHAL SAMPLING METHOD FOR THE DETECTION OF Enterocytozoon hepatopenaei IN SHRIMP. Aquaculture America- World Aquaculture Society Meeting. New Orleans, LA.
• Cruz-Flores, R., Mai, H., Aranguren Caro, L. F., Millabas, J., Kaneshamoorthy, S., & Dhar, A. K. (2019, February). PATHOGEN DETECTION FROM DAVIDSON´S FIXED PARAFFIN EMBEDDED SHRIMP TISSUE. Aquaculture America- World Aquaculture Society Meeting. New Orleans, LA.
• Cruz-Flores, R., Mai, H., Kanrar, S., Aranguren caro, L. F., & Dhar, A. K. (2019, November). COMPLETE GENOME RECONSTRUCTION OF WHITE SPOT SYNDROME VIRUS FROM ARCHIVAL DAVIDSON-FIXED PARRAFFIN EMBEDDED SHRIMP TISSUE. Latin America & Caribbean Aquaculture Conference (LAQUA)- World Aquaculture Society Meeting.
• Dhar, A. K., Aranguren Caro, L. F., Mai, H., Kanrar, S., & Cruz-Flores, R. (2019, July). Characterization of a novel mutant of Vibrio parahaemolyticus that carries binary toxin genes, pirA and pirB but does not cause acute hepatopancreatic necrosis disease (AHPND) in Pacific white shrimp (Penaeus vannamei).. Annual Meeting of the Society of Invertebrate Pathologists. Valencia, Spain.
• Dhar, A. K., Cruz-Flores, R., Blaylock, R., Curran, S., & Allnutt, F. C. (2019, June). Can White Spot Disease Control be Achieved Using CRISPR/Cas Driven Genome Editing in Live Shrimp?. Asia Pacific- World Aquaculture Society Meeting. Chennai, India.
• Dhar, A. K., Garfias, M., Millabas, J., Lin, J., Lyons, G., Cruz-Flores, R., Mai, H., Kanrar, S., & Aranguren Caro, L. F. (2019, February). Evolving Trends in Shrimp Disease Diagnostics. Aquaculture America, World Aquaculture Society Meeting. New Orleans, LA.
• Noble, B., Cruz-Flores, R., Lin, J., & Aranguren Caro, L. F. (2019, February). COMPARATIVE STUDY OF Enterocytozoon hepatopenaei (EHP) CHALLENGE METHODS IN Penaeus vannamei. Aquaculture America- World Aquaculture Society Meeting. New Orleans, LA.
• Schofield, P., Aranguren Caro, L. F., Noble, B., Padilla, T., Mai, H., Millabas, J., & Dhar, A. (2019, February). INVESTIGATION INTO THE IMPACT OF ACUTE HEPATOPANCREATIC NECROSIS DISEASE (AHPND) ON THE FRESHWATER PRAWN Macrobrachium rosenbergii and PACIFIC WHITE SHRIMP (Penaeus vannamei) AT VARIOUS SALINITIES. Aquaculture America- World Aquaculture Society Meeting. New Orleans, LA.
• Dhar, A. K. (2018, Fall). Aquaculture Pathology Laboratory.. World Organization for Animal Health (OIE, Paris, France) Twinning Project Workshop, Jeddah Fish Health Safety Laboratory, Jeddah, Saudi Arabia, November 04, 2018..
• Dhar, A. K. (2018, Fall). The University of Arizona, Aquaculture Pathology Laboratory: A Worldwide Resource for Diagnostic Services and Collaborative Research to Shrimp Aquaculture Industry.. 8th International Society of Aquatic Animal Health Conference, Charlottetown, PEI, Canada, Sept 03-06, 2018..
• Dhar, A. K. (2018, Fall). “Biosecurity & Health in US Indoor Shrimp Farming”. Kentucky State University, Indoor Shrimp Farming Workshop, hosted by Kentucky State University (KSU), September 14-15, 2018..
• Dhar, A. K. (2018, Fall). “Current status of acute hepatopancreatic necrosis disease in shrimp: biology, diagnostics and disease management”, International Society of Aquatic Animal Health, Charlottetown, PEI, Canada, Sept 03-06, 2018.. 8th International Society of Aquatic Animal Health Conference, Charlottetown, PEI, Canada, Sept 03-06, 2018..
• Dhar, A. K. (2018, Fall). “Overview of shrimp diseases worldwide”.. OIE Twinning Project Workshop, Jeddah Fish Health Safety Laboratory, Jeddah, Saudi Arabia, November 04, 2018..
• Dhar, A. K. (2018, Fall). “The current status of knowledge on viral disease and its impact for commerce".. 10th International Symposium on Aquatic Animal Health and Food Safety, September 18, 2018, Ensenada, Mexico..
• Dhar, A. K. (2018, Spring). “Acute Hepatopancreatic Necrosis Disease (AHPND) and Entercytozoon hepatopenaei (EHP): Two emerging threats to shrimp aquaculture”. AQUAINDIA-2018, Chennai, India, Feb 02-03, 2018.
• Dhar, A. K. (2018, Summer). AHPND and EHP and Their Control in Shrimp Farming”. Guatemala Aquaculture Symposium, Antigua, Guatemala, June 06-08, 2018.

Others

• Aranguren Caro, L. F., Mai, H., Pichardo, O., Hanggono, B., & Dhar, A. K. (2019, May). White Feces Syndrome in shrimp: Predictor of EHP?. Global Aquaculture Advocate.