Rita M. Redman

Shrimp diseases and current diagnostic methods

Abstract In less than 30 yr, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of U.S. dollars in revenue, and augmentation of the worlds food supply with a high value crop. Concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. Major epizootics have plagued the worlds shrimp culture industries. The most important diseases of cultured penaeid shrimp have had viral or bacterial etiologies, but a few important diseases have fungal and protozoan agents as their cause. Diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. While tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. The need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. The industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like IHHNV, HPV, TSV, WSSV, MBV, and BP. Additional genomic probes for viruses, for bacterial pathogens like NHP and certain Vibrio spp., and Microsporidia have also been developed. Highly sensitive detection methods for some pathogens that employ DNA amplification methods based on the polymerase chain reaction (PCR) now exist, and more PCR methods are being developed for additional agents. These advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease.

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.

Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp.

Abstract Populations of the Pacific blue shrimp, Penaeus stylirostris, reared at the University of Arizonas experimental shrimp culture facility on Oahu in Hawaii from late 1980 through 1981, were severely affected by a highly acute and lethal disease of viral etiology. Also found to be susceptible to the disease were P. vannamei and P. monodon. The disease was named infectious hypodermal and hematopoietic necrosis (IHHN) disease to describe the principal lesions observed. The histopathology of acute and subacute IHHN disease in these species was dominated by the presence of conspicuous eosinophilic intranuclear-inclusion bodies of the Cowdry type A variety in ectodermally (especially the cuticular hypodermis) and mesodermally (especially the hematopoietic tissues) derived tissues that were undergoing necrosis. Electron microscopy of affected tissues demonstrated the presence of two or three types of virus-like particles with cubic morphology and diameters of 17 to 27 nm that suggest IHHN virus to be either a parvo- or picornavirus.

Experimental Infection of Western Hemisphere Penaeid Shrimp with Asian White Spot Syndrome Virus and Asian Yellow Head Virus

Abstract Postlarval and juvenile stages of four species of western hemisphere penaeid shrimp (Penaeus aztecus, P. duorarum, P. setiferus, and P. vannamei) were experimentally challenged with white spot syndrome virus (WSSV) and yellow head virus (YHV) isolates originating from Asia. Challenge exposures were accomplished by feeding minced tissue from WSSV- or YHV-infected shrimp tissues. The WSSV challenge of postlarval shrimp resulted in severe infections in P. setiferus and P. vannamei and less severe infections in P. aztecus and P. duorarum. The WSSV challenge of juvenile shrimp (∼1 g) resulted in severe infections and 100% cumulative mortality in P. setiferus and P. vannamei, moderate infections and 27% cumulative mortality in P. aztecus, and no signs of infection and 0% cumulative mortality in P. duorarum. The YHV challenge caused serious disease and mortality in juveniles of all four species, but postlarval shrimp appeared resistant to YHV because no virus-related signs of infection, mortality, or di...

Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. vannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. vannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35–60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host.

A parvo-like virus disease of penaeid shrimp

Abstract Cultured populations of four penaeid shrimp species (Crustacea, Decapoda) from four separate culture facilities in Asia were found to be adversely affected by a disease of presumed viral etiology. Individual shrimp with the disease displayed nonspecific signs, including poor growth rate, anorexia, reduced preening activity, increased surface fouling, and occasional opacity of tail musculature. These signs were accompanied by mortalities during the juvenile stages, after apparently normal development through the larval and postlarval stages. Accumulative mortality rates in epizootics in Penaeus merguiensis and P. semisulcatus reached as high as 50 to 100%, respectively, of the affected populations within 4 to 8 weeks of disease onset. The principal lesion, common to all four species, was necrosis and atrophy of the hepatopancreas, accompanied by the presence of large prominent basophilic, PAS-negative, Fuelgen-positive intranuclear inclusion bodies in affected hepatopancreatic tubule epithelial cells (hepatopancreatocytes). These inclusion bodies presumably developed from small, eosinophilic, intranuclear bodies that were also present in the affected tissues. Electron microscopy of affected hepatopancreatocytes revealed aggregations of 22- to 24-nm-diameter virus particles within the electron-dense granular inclusion body ground substance. The virus particle size and morphology, the close association of the nucleolus with the developing inclusion body, and the presence of intranuclear bodies within developing inclusion bodies are similar to cytopathological features reported for parvovirus infections in insects and vertebrates. It is suggested that this presumed virus disease of cultured penaeid shrimp be called HPV for Hepatopancreatic Parvo-like Virus disease.

Observations on the geographic distribution, pathogenesis and morphology of the baculovirus from Penaeus monodon Fabricius

Abstract A baculovirus disease of Penaeus monodon Fabricius named monodon baculovirus (MBV) was found in populations of postlarval P. monodon that originated from three separate sources in the Southwestern Pacific region. The pathogenesis, lesion development and morphology of the virus was the same regardless of the origin of the P. monodon population. The apparent target organs and tissues for MBV are the hepatopancreatic tubule and duct epithelium of postlarvae, juveniles and adults, and the anterior midgut epithelium of very young postlarvae. It was not determined if MBV produces patent disease in the larval stages of P. monodon , nor how it is transferred from brood stock to their offspring. MBV disease may cause serious losses in compromised populations (especially in postlarvae and young juveniles), but under conditions favorable to the host, fewer mortalities occurred and only low-grade infections of the hepatopancreas were detected. Three stages in the pathogenesis of MBV disease were noted in the hepatopancreas. Stage 1 hepatopancreatocytes had slightly hypertrophied nuclei, but contained no occlusion bodies (a highly specialized type of inclusion body) and few completed virions; stage 2 cell nuclei also had hypertrophied nuclei, but contained developing occlusion bodies and completed virions; stage 3 cell nuclei contained mature occulusion bodies and abundant numbers of completed free and occluded virions. Cell necrosis and cytolysis or sloughing (releasing virus and occlusion bodies into the gut lumen) followed stage 3 development in living hosts. The morphology of MBV is consistent with members of the genus Baculovirus subgroup A. The MBV particles are rod-shaped, singly enveloped, and replicate in the nucleus, occur free or within proteinaceous polyhedral occlusion bodies, and are assumed to contain DNA as the nucleic acid type. The nucleocapsid of MBV measured 42 ± 3 nm by 246 ± 15 nm, while the enveloped virions measured 75 ± 4 nm by 324 ± 33 nm.

A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes

In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidsons AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidsons fixative, which is highly acidic (pH approximately 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidsons fixative or a new, near neutral (pH approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidsons preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidsons, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.

Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV

There is currently concern and controversy in the shrimp industries of the Americas about the risk posed by the importation and reprocessing of shrimp infected with white spot syndrome virus (WSSV) and yellow head virus (YHV). To further understand the risk, more knowledge concerning the quantitative virus load of infected shrimp is needed. The present study was carried out to better define, using qualitative and quantitative methods, the relative virus load of shrimp heads and tails. For these studies, specific pathogen-free (SPF) Penaeus vannamei were infected with WSSV. Emergency harvest of these shrimp was simulated by collecting the infected shrimp at the onset of postinfection mortalities and determining the relative virus loads of the head and tails by quantitative real-time PCR and histology methods. Routine histology and in situ hybridization assay with a WSSV-specific DNA probe demonstrated qualitatively similar levels of WSSV infection in the heads and tails of experimental infected shrimp. The novel real-time PCR method demonstrated quantitatively that the head had a slightly higher WSSV load than did the tail. However, since the tail represents 58% of the total body weight, the total virus load on a per weight basis turns out to be similar in the head (49%) and tail (51%) of the same shrimp with acute phase WSSV infections. In proportion to the total tail weight, the virus load of the peeled shell represents 55% of the total viral load in tail.

The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV-specific cDNA probes

Abstract Representative archived Litopenaeus vannamei samples (117 total), originating from 13 different countries and collected between 1992 to 1996, were analyzed by in situ hybridization to verify the presence of Taura Syndrome Virus (TSV) within pathodiagnostic acute phase TS histological lesions. The in situ assay results showed that TSV was present in one or more representative samples analyzed from each country (76 of 117 samples or 65%), thus, confirming the original histological diagnosis of TSV infection. The false negative in situ hybridization results, obtained for 35% of the samples assayed (41 in total), were attributed to over-fixation with Davidsons AFA (acetic acid, formaldehyde, alcohol) solution and consequent acid hydrolysis of TSV genomic RNA within pathodiagnostic TSV lesions. The collective findings of this disease survey assisted in documenting the spread and current distribution of TSV over a 5-year period and definitively established the presence of TSV within TS diseased shrimp originating from Ecuador when and where the disease was first recognized in 1992. These findings further strengthen the existing evidence that TS has a viral, not a toxic, etiology and indicate that either a single TSV strain, or very similar strains of the same virus, are responsible for the TSV panzootic that has been expanding in the Americas since 1992.