Fernando Mendoza-Cano

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

BackgroundThe White Spot Syndrome Virus (WSSV), the sole member of the family Whispoviridae, is the etiological agent that causes severe mortality events in wild and farmed shrimp globally. Given its adverse effects, the WSSV has been included in the list of notifiable diseases of the Office of International Epizootic (OIE) since 1997. To date there are no known therapeutic treatments available against this lethal virus, and a surveillance program in brood-stock and larvae, based on appropriate diagnostic tests, has been strongly recommended. However, some currently used procedures intended for diagnosis of WSSV may be particularly susceptible to generate spurious results harmfully impacting the shrimp farming industry.MethodsIn this study, a sensitive one-step SYBR green-based real-time PCR (qPCR) for the detection and quantitation of WSSV was developed. The method was tested against several WSSV infected crustacean species and on samples that were previously diagnosed as being positive for WSSV from different geographical locations.ResultsA universal primer set for targeting the WSSV VP28 gene was designed. This method demonstrated its specificity and sensitivity for detection of WSSV, with detection limits of 12 copies per sample, comparable with the results obtained by other protocols. Furthermore, the primers designed in the present study were shown to exclusively amplify the targeted WSSV VP28 fragment, and successfully detected the virus in different samples regardless of their geographical origin. In addition, the presence of WSSV in several species of crustaceans, including both naturally and experimentally infected, were successfully detected by this method.ConclusionThe designed qPCR assay here is highly specific and displayed high sensitivity. Furthermore, this assay is universal as it allows the detection of WSSV from different geographic locations and in several crustacean species that may serve as potential vectors. Clearly, in many low-income import-dependent nations, where the growth of shrimp farming industries has been impressive, there is a demand for cost-effective diagnostic tools. This study may become an alternative molecular tool for a less expensive, rapid and efficient detection of WSSV.

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) is a single-stranded DNA virus that infects several penaeid shrimp species, provoking economic losses in farmed shrimp populations estimated at several million of dollars. Furthermore, IHHNV has historically been considered an important threat for wild shrimp populations, but its real measurable impact remains unknown. Currently no treatments are available against IHHNV, and research to develop potential antiviral strategies depends on a detailed understanding of the viral life cycle. However, the exact pathophysiological events underlying the development of metabolic changes in IHHNV-infected shrimp are still unknown. Thus, the biochemical changes caused by the IHHNV infection in plasma and hepatopancreas of the economically important shrimp species Penaeus vannamei were evaluated. Glucose, lactate, total protein, glycogen, triacylglycerides, cholesterol, and total lipids were measured in healthy and IHHNV-infected shrimp. Significant changes were observed in energy substrates (glucose, lactate, triacylglycerides and cholesterol), in plasma and hepatopancreas. These changes may indicate a temporal sequestration of the host-cell metabolic pathways by the virus to maximize its replication and propagation.

Evaluation of the productive and physiological responses of Litopenaeus vannamei infected with WSSV and fed diets enriched with Dunaliella sp.

An experimental investigation was performed to evaluate the productive and physiological responses of Litopenaeus vannamei that were infected with WSSV and fed diets enriched with Dunaliella sp., which had a high β-carotene content induced by nitrogen reduction in the culture medium. A basal diet containing 35% crude protein and experimental diets T1 and T2, which included 1% and 2% microalgae meal, respectively, were evaluated. Positive (infected juveniles) and negative (non-infected) controls were also evaluated. Survival was significantly higher (80%) in the two treatment groups compared to the positive control group (56%). In the negative control group, survival was 100%. Some variation was recorded for hemolymph metabolites among treatments and at distinct times post-infection; although the tendencies were not clear, some metabolites (glucose and triglycerides) appeared to decrease on the last days of the trial, probably due to their use as energy for the shrimp to thrive despite the infection. The results of the study suggest a positive effect of the dietary inclusion of Dunaliella meal on shrimp survival and an unclear effect on hemolymph metabolites.

An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp

The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

Prevalence of the infectious hypodermal and hematopoietic necrosis virus in shrimp (Penaeus vannamei) broodstock in northwestern Mexico

The Penaeus stylirostris densovirus (PstDNV or IHHNV) is the smallest of the known shrimp viruses. It causes severe mortalities in juveniles and sub-adults of the blue shrimp Penaeus stylirostris, while specimens of the white shrimp Penaeus vannamei infected by this virus exhibit reduced growth rates and negative effects on the feed-conversion rate (FCR). To date, no descriptive epidemiological surveys on the prevalence of this virus in shrimp broodstock have been performed. In this study, the prevalence of IHHNV in broodstock of the white shrimp P. vannamei from hatcheries on the northwest of Mexico region was estimated. Prevalence vary across different regions from high (63%) to low (6%) in shrimp broodstock. Several factors, as transport of pathogens by human activities, or the absence or implementation of ineffective biosecurity measures, may explain the observed differences. To the best of our knowledge, the present study is the first to examine the prevalence of IHHNV on broodstock.

The Endemic Copepod Calanus pacificus californicus as a Potential Vector of White Spot Syndrome Virus

The susceptibility of the endemic copepod Calanus pacificus californicus to white spot syndrome virus (WSSV) was established by the temporal analysis of WSSV VP28 transcripts by quantitative real-time PCR (qRT-PCR). The copepods were collected from a shrimp pond located in Bahia de Kino Sonora, Mexico, and challenged per os with WSSV by a virus-phytoplankton adhesion route. Samples were collected at 0, 24, 48 and 84 h postinoculation (hpi). The VP28 transcripts were not detected at early stages (0 and 24 hpi); however, some transcript accumulation was observed at 48 hpi and gradually increased until 84 hpi. Thus, these results clearly show that the copepod C. pacificus californicus is susceptible to WSSV infection and that it may be a potential vector for the dispersal of WSSV. However, further studies are still needed to correlate the epidemiological outbreaks of WSSV with the presence of copepods in shrimp ponds.

Selection and validation of candidate reference genes for quantitative real-time PCR studies in the shrimp Penaeus vannamei under viral infection

The decapod Penstyldensovirus 1 (PstDV-1) represents one of the most serious threats for penaeid shrimp farming. Studies aimed at defining relevant molecular effects of this virus over its host are imperative in the attempt to increase our understanding of its pathogenesis. Unfortunately, few studies have focused on the definition of the expression profile of reference genes in shrimp challenged with a pathogen. As a result, there are no studies on the selection of reference genes for the normalization of target gene expression changes yielding reliable data of the effects following PstDV-1 infection in shrimp. Therefore, the aim of the present study was to evaluate and validate the appropriateness of four candidate reference genes (ef1-α, gapdh, rpl8 and β-tubulin) for their use as reference genes to normalize qPCR data in gene expression studies of PstDV-1-shrimp interactions. By analyzing the expression profile of those genes, gapdh was validated as a suitable reference gene to normalize expression data gathered from a PstDV1-challenge, while ef1-α, β-tubulin, and rpl8 were identified as unstably expressed during the infectious process. The suitability of gapdh as a common reference gene in studies of host gene response to viral infections is underlined.

Marine Viruses: the Beneficial Side of a Threat

Marine viruses are ubiquitous, extremely diverse, and outnumber any form of life in the sea. Despite their ecological importance, viruses in marine environments have been largely ignored by the academic community, and only those that have caused substantial economic losses have received more attention. Fortunately, our current understanding on marine viruses has advanced considerably during the last decades. These advances have opened new and exciting research opportunities as several unique structural and genetic characteristics of marine viruses have shown to possess an immense potential for various biotechnological applications. Here, a condensed overview of the possibilities of using the enormous potential offered by marine viruses to develop innovative products in industries as pharmaceuticals, environmental remediation, cosmetics, material sciences, and several others, is presented. The importance of marine viruses to biotechnology should not be underestimated.

Experimental Infection and Detection of Necrotizing Hepatopancreatitis Bacterium in the American Lobster Homarus americanus

Necrotizing hepatopancreatitis bacterium (NHPB) is an obligated intracellular bacteria causing severe hepatopancreatic damages and mass mortalities in penaeid shrimp. The worldwide distribution of penaeid shrimp as alien species threatens the life cycle of other crustacean species. The aim of the experiment was to evaluate the possibility of experimentally infecting the American lobster (Homarus americanus) with NHPB extracted from shrimp hepatopancreas. Homogenates from infected shrimp were fed by force to lobsters. Other group of lobsters was fed with homogenates of NHPB-free hepatopancreas. After the 15th day from initial inoculation, the presence of NHPB was detected by polymerase chain reaction in feces and hepatopancreas from lobsters inoculated with infected homogenates. Necrotized spots were observed in the surface of lobster hepatopancreas. In contrast, lobsters fed on NHPB-free homogenates resulted negative for NHPB. Evidence suggests the plasticity of NHPB which can infect crustacean from different species and inhabiting diverse latitudes. Considering the results, the American lobster could be a good candidate to maintain available NHPB in vivo.

Molecular epidemiology of selected infectious diseases caused by bacteria in juveniles and post‐larvae of the white shrimp Penaeus vannamei from the north‐western coast of Mexico

Over the past decades, shrimp farming has become a rapidly expanding industry in many countries, particularly in Asia and South America. In 2000, the production of cultured shrimp reached 27.3% of the total volume (Norhana et al. 2010), but in 2014, the amount of farmed shrimp surpassed for the first time, that of the wild fisheries (Scarrat 2014). Unfortunately, this impressive development has been accompanied by some practices that promote the development of stress conditions that contribute to the appearance of opportunistic pathogens. Hence, bacterial diseases are a major cause of growth retardation and mass mortalities in shrimp farming (Flegel 2012). The major, and perhaps most common, bacterial diseases currently affecting shrimp farming are vibriosis and necrotizing hepatopancreatitis bacterium (NHP-B) (Vincent & Lotz 2005). Moreover, an emerging disease designated as acute hepatopancreatic necrosis disease (AHPND), caused by a strain of the bacteria Vibrio parahaemolyticus, has been reported to cause significant losses among shrimp farms (Tran et al. 2013). The state of Sonora led the overall production of shrimp (aquaculture and trawling combined) in Mexico for several years. However, in the following years, the production declined due to the presence of the white spot syndrome virus (WSSV). Furthermore, in 2013, shrimp farms from the north-western coast of Mexico were affected by atypical mortalities that occurred few days after stocking which were associated with the presence of AHPND (hereafter referred to as VPAHPND) (Nunan et al. 2014; Soto-Rodriguez et al. 2015), although uncertainty regarding its prevalence remains among shrimp farming producers. The prevalence of the necrotizing hepatopancreatitis bacterium (NHP-B), V. parahaemolyticus and VPAHPND was estimated in juveniles of the Penaeus vannamei from aquaculture facilities on the north-western coast of Mexico, and a set of pooled samples was made from five specimens. A total of 54 pooled samples (n = 5) were formed. Samples were arbitrarily collected during the 2014–2015 shrimp farming season at commercial farms located in north-western Mexico, essentially in the states of Sonora and Sinaloa. In addition, the prevalence of these pathogens in shrimp postlarvae (PL) was estimated in a total of 192 PL. Shrimp and PL samples were fixed with 70% alcohol. PL were randomly collected from 16 different commercial hatcheries identified by letters A through P (12 PL from each hatchery). The surveys were conducted during 2014 in hatcheries Correspondence Dr. A S anchez Paz, Centro de Investigaciones Biol ogicas del Noroeste S. C. Laboratorio de Referencia, An alisis y Diagn ostico en Sanidad Acu ıcola, Hermosa 101, Col. Los Angeles, Hermosillo, Sonora C. P. 83106, M exico (e-mail: asanchez04@cibnor.mx)