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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., Graphical abstract Image 1, Highlights • Development of a multiplex SYBR Green real-time PCR for the simultaneous detection of the pirA and pirB genes of Vibrio spp. • Comparison of the SYBR Green assay with the TaqMan assay for the detection of the pirA and pirB genes of Vibrio spp. • First report of real-time PCR assays for the simultaneous detection of the pirA and pirB genes of Vibrio spp.

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.

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.

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.

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.

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 PirAVp and PirBVp. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (VpAHPND) 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 VpAHPND 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 pirAVp and pirBVp 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 pirAVp and pirBVp 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.

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.

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 PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (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 PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp 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 PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.

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 (VPAHPND). 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 VPAHPND 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 VPAHPND 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.

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.

The propagation of Taura syndrome virus (TSV) in primary hemocyte culture of Pacific white shrimp (Penaeus vannamei) was investigated. Purified TSV was inoculated into a 24 h old primary hemocyte culture and the development of cytopathic effects was monitored. The cell morphology started changing within 6 h post-inoculation; TSV-infected hemocytes started shrinking and granular structures began to form on the cell surface. There was a gradual loss of cell viability and, by 48 h post-inoculation, most cells detached from the bottom of the 96 well microplate. The propagation of TSV during the 48 h time course studied was measured by real-time RT-PCR. TSV copy number reached the highest level by 12 h post-inoculation and then started to decrease. Using an anti-TSV polyclonal antibody, the 55 kDa VP1 capsid protein was detected by Western blot analysis. The data suggest that shrimp primary hemocyte culture supports TSV replication and could be used as a tool for the study of host-virus interactions in TSV pathogenesis.

Infectious pancreatic necrosis virus (IPNV) is a major viral pathogen of salmonid fish and causes serious economic losses to salmonid aquaculture. Previously, we demonstrated that the IPNV capsid protein, VP2, expressed in yeast self-assembles into subviral particles (SVPs) and injection of these IPNV rVP2 SVPs into rainbow trout elicits an immune response. Immunized fish had reduced viral loads compared to unimmunized fish when challenged with IPNV. To evaluate the suitability of IPNV rVP2 SVPs for future development of multivalent vaccines, a linear epitope of a human oncogene, c-myc, was cloned into the IPNV rVP2 SVP backbone as a model epitope and expressed in yeast. Western blot analyses with anti-c-myc and anti-IPNV antibodies provided positive identification of both the c-myc and VP2 epitopes on the c-myc VP2 SVPs. Transmission electron microscopy of purified chimeric c-myc VP2 SVPs revealed the formation of approximately 20nm particles. Rainbow trout immunized with c-myc VP2 SVPs elicited both anti-c-myc and anti-IPNV immune responses. When immunized fish were challenged with IPNV, the viral load in the c-myc VP2 SVP immunized fish was significantly lower than the sham-vaccinated controls. The results indicate that IPNV rVP2 SVPs can tolerate the insertion of foreign epitopes without affecting either the antigenic potential of the epitopes of the backbone protein or the inserted foreign epitope. This opens the possibility of using the IPNV rVP2 SVP platform to express epitopes of other viruses, which could pave the way for development of multivalent subunit vaccines or novel marker vaccines.

In order to assess the microbial water quality of the lagoon canals of Venice, Italy and nearby beach on Lido island, a study was conducted using real-time RT-PCR to enumerate levels of hepatitis A virus (HAV) and enteroviruses in these marine waters over a 3-year period from 2003 to 2005. A total of 17 sites (9 lagoon canal and 8 beach sites) were assayed. For the canals of the Venice Lagoon, 78% were positive for both HAV and enteroviruses, with levels ranging from 75 to 730 and 3 to 1,614 genome copies/L, respectively. At Lido beach, HAV was never detected, but enteroviruses were detected in all Lido beach samples at levels ranging from 2 to 71 genome copies/L. There was a statistically significant correlation between thermotolerant coliform densities and HAV levels (p=0.0002), but the relationship between thermotolerant coliform densities and enterovirus levels was not significant (p>0.05). Despite the fact that enteroviruses were detected at low levels in the surfzone at Lido beach, the risk for enteroviral infection (calculated using the beta-Poisson model) for recreational exposure from swimming, was in the range of 1.9 x 10(-3) - 6.1 x 10(-2), yielding a disease risk at or below the level (5% for gastroenteritis) deemed acceptable by European Guide standards.

Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that binds to double stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the DNA. The indiscriminate binding ability of SYBR Green I dye to dsDNA often results in non-specific products. We have shown that increasing the amplicon size from approximately 50 to approximately 75-100 bp increases the specificity due to higher melting temperature of the amplicon and also enhances the sensitivity of detection of real-time RT-PCR using SYBR Green chemistry while detecting two RNA viruses in laboratory-challenged shrimp, the Taura syndrome virus (TSV), and yellow head virus (YHV). The increased sensitivity of the larger amplicon over the smaller amplicon varied from 1.6 to 6.82-fold (with a median value of 4-fold) for the TSV-infected samples, and 1.80-10.27-fold (with a median value of 4-fold) for the YHV-infected samples. The longer amplicon also has a higher Tm value compared with the shorter amplicon (75.6 vs. 72.0 degrees C for TSV, and 81.3 vs. 72.5 degrees C for YHV). The increased melting temperature of the longer amplicon compared with the shorter amplicon will enable easier discrimination of a specific product from a primer dimer or other non-specific products. The improved method for the detection of TSV and YHV will be applicable not only to the detection of other viral pathogens but also to the quantitative measurement of cellular gene expression by real-time SYBR Green RT-PCR.

Taura syndrome virus (TSV) and yellow head virus (YHV) are the two RNA viruses infecting penaeid shrimp (Penaeus sp.) that have caused major economic losses to shrimp aquaculture. A rapid and highly sensitive detection and quantification method for TSV and YHV was developed using the GeneAmp 5700 Sequence Detection System and SYBR Green chemistry. The reverse transcriptase polymerase chain reaction (RT-PCR) mixture contained a fluorescent dye, SYBR Green, which exhibits fluorescence enhancement upon binding to double strand cDNA. The enhancement of fluorescence was found to be proportional to the initial concentration of the template cDNA. A linear relationship was observed between input plasmid DNA and cycle threshold (C(T)) values for 10(6) down to a single copy of both viruses. To control for the variation in sample processing and in reverse transcription reaction among samples, shrimp beta-actin and elongation factor-1alpha (EF-1alpha) genes were amplified in parallel with the viral cDNA. The sensitivity and the efficiency of amplification of EF-1alpha was greater than beta-actin when compared to TSV and YHV amplification efficiency suggesting that EF-1alpha is a better internal control for the RT-PCR detection of TSV and YHV. In addition, sample to sample variation in EF-1alpha C(T) value was lower than the variation in beta-actin C(T) value of the corresponding samples. The specificity of TSV, YHV, EF-1alpha and beta-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. The C(T) values of TSV and YHV samples were normalized against EF-1alpha C(T) values for determining the absolute copy number from the standard curve of the corresponding virus. The method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture.

We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).

Offspring of four crosses (I, II, III, and IV) ofPenaeus vannameifrom known high- and low-growth families were challenged with infectious hypodermal and hematopoetic necrosis virus (IHHNV) andBaculovirus penaei(BP) to compare their susceptibility to these viral agents and examine the genetic component involved in disease resistance or susceptibility. Family crosses were made using broodstock from five families developed by the U.S. Marine Shrimp Farming Program. The prevalence of IHHNV infection was highest in cross I and lowest in cross III. Cross I was developed using male and female broodstock from the low-growth family 1.6, and cross III was developed using a female from the high-growth family 1.3 and a male from the low-growth family 1.6. The prevalence of BP infection at Day 4 was highest (100%) in cross IV, which was developed using a female from the low-growth family 1.4 and a male from the high-growth family 1.5. The reciprocal cross, cross III, had the lowest (68%) prevalence at Day 4 postexposure. Both crosses I and II had 88% prevalence of infection at Day 4. Despite 100% prevalence of BP infection in cross IV at 4 days, animals from this cross and cross II exhibited high survival by Day 18 (85 and 77%). On the other hand, crosses I and III (with 88 and 68% prevalence at Day 4, respectively) showed low survival at Day 18 (19 and 24%). On the basis of prevalence of infection and mortality rates, it was concluded that the susceptibility to BP in penaeid shrimp is governed by the genetic background of the parental crosses. The random amplified polymorphic DNA polymorphisms for crosses I, II, III, and IV, were 43, 45, 53, and 51%, respectively, showing no clear relationship between IHHNV and BP prevalence of infection and levels of nuclear genetic diversity. Though the mtDNA haplotypes in offspring from the different crosses were the same, major differences were observed in both steady-state levels and patterns of expression of the mitochondrial 12s rRNA in offspring obtained at various early developmental stages from each of the four crosses. The possible relationship among disease susceptibility, growth status, and expression of mitochondrial 12s rRNA is discussed in the context of a complex nuclear–cytoplasmic genetic system involved in the regulation of gene expression.