Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses in hatchery-reared Penaeus monodon postlarvae

The prevalence of hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in samples of Penaeus monodon postlarvae (PL10 to PL20, 10 to 20 d old postlarvae) in India was studied by PCR. Samples collected from different hatcheries, and also samples submitted by farmers from different coastal states, were analyzed. HPV was detected in 34%) of the hatchery samples and 31% of the samples submitted by farmers, using a primer set designed for detection of HPV from P. monodon in Thailand. However, none of these samples were positive using primers designed for detection of HPV from P. chinensis in Korea. This indicated that HPV from India was more closely related to HPV from P. monodon in Thailand. MBV was detected in 64% of the samples submitted by the farmers and 71% of the hatchery samples. A total of 84 % of the samples submitted by farmers, and 91% of the hatchery samples, were found positive for WSSV. Prevalence of concurrent infections by HPV, MBV and WSSV was 27% in hatchery samples and 29%, in samples submitted by farmers. Only 8% of the hatchery samples and 16% of the samples submitted by farmers were negative for all 3 viruses. This is the first report on the prevalence of HPV in P. monodon postlarvae from India.     

Detection and quantification of hepatopancreatic parvovirus in penaeid shrimp by real-time PCR assay

As one of the major pathogens, hepatopancreatic parvovirus (HPV) can cause severe diseases in penaeid shrimp. We developed a TaqMan-based real-time PCR assay for the HPV detection in China. A pair of primers (HPVF and HPVR) and a TaqMan probe were designed according to the HPV genomic sequence of Chinese isolate (GenBank: GU371276). Our data showed that the primers and TaqMan probe were specific for HPV, and they exhibited no cross-reaction with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and specific pathogen free (SPF) shrimp DNA. The assay had a detection limit of four plasmid HPV DNA copies per reaction. Furthermore, HPV was detected in 16 of 21 Fenneropenaeus Chinensis, 3 of 52 Litopenaeus vannamei and 2 of 2 Marsupenaeus japonicus penaeid shrimp samples. In addition, HPV was also detected in crabs. Therefore, this assay could be successfully used as a sensitive and rapid molecular-based diagnostic method to screen HPV-free animals and survey the prevalence of HPV in cultured populations of penaeid shrimp in China.     

Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.     

Occurrence, histopathology and experimental transmission of hepatopancreatic parvovirus infection in Penaeus monodon postlarvae

Hepatopancreatic parvovirus (HPV) was detected in samples of Penaeus monodon post-larvae (PL-13, PL-18, PL-19, PL-26) from 2 hatcheries in 2 provinces (Samar and Iloilo) in the Philippines. The percentage of infection was 20 to 100% in postlarvae obtained from the hatchery in Samar in August 2001. Postlarvae from the hatchery in Iloilo, sampled in October and November 2001, had 70 to 99% HPV infection. Wet mounts of squashed hepatopancreatic tissue stained with malachite green (wet-mount technique) and histopathology revealed the presence of large, usually single, basophilic intranuclear inclusion bodies in the distal tubules, which led to displacement of the nucleoli. Light microscopy showed ovoid to spherical inclusion bodies, 5 to 11 mmicrom in diameter. Transmission electron microscopy revealed that the inclusion bodies were composed of electron-dense granular material and virions. The virions appeared roughly spherical and averaged 18 to 22 nm in diameter. An experiment was undertaken to induce HPV infection by feeding P. monodon postlarvae with virus-infected postlarvae. P. monodon postlarvae (PL-16), initially determined as free from HPV, were found HPV-positive 24 h after being fed with infected material. The percentage of infection ranged from 30% at Day 1 post-infection (p.i.) to 100% at Day 7 p.i. determined by the wet-mount technique and by histopathology. This is the first report of a successful horizontal transmission of HPV in P. monodon postlarvae.     

Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.     

PCR结合反向斑点杂交法检测石蜡包埋组织中的曲霉感染

目的评价PCR结合反向斑点杂交法检测福尔马林固定、石蜡包埋组织中曲霉感染的可行性。方法选取39例病理证实曲霉感染的患者活检标本（21例为鼻窦感染标本、18例为尸检标本）,以1对真菌特有的28SrRNA保守序列结构作为真菌通用引物,以临床常见的4个曲霉菌种：烟曲霉、黄曲霉、黑曲霉、土曲霉的种特异性序列为种特异性探针,与扩增产物进行反向斑点杂交。结果尸检标本阳性率为55．6％（10／18）,鼻窦标本阳性率为76．2％（16／21）,特异性均为100％。在这些曲霉所致的系统性感染中,烟曲霉是主要的致病真菌。结论该方法能对临床无法培养的石蜡组织块进行回顾性病原学研究,并可以鉴定常见的曲霉菌种,有良好的特异性和敏感性,适用于临床曲霉感染的检测。     

PCR结合反向斑点杂交法检测侵袭性曲霉感染的初步实验研究

目的评价PCR结合反向斑点杂交法检测动物模型和临床免疫抑制患者的侵袭性曲霉感染的可行性。方法建立侵袭性曲霉感染动物模型,收集动物血清和健康人群、免疫抑制患者的血清,进行PCR-种特异性探针检测。以1对真菌特有的28S rRNA保守序列结构作为真菌通用引物,以临床常见的4个曲霉菌种,即烟曲霉、黄曲霉、黑曲霉、土曲霉的种特异性序列为种特异性探针,与扩增产物进行反向斑点杂交。结果PCR-探针杂交检测动物接种后24h时阳性血清为16/24,48h为18/24,72h为19/24,96h为24/24。阳性率为80%,特异性为95.8%。同时取血行真菌培养,烟曲霉阳性率仅为5%。临床标本显示两例确诊IA患者血清均为阳性,且能鉴定菌种。健康对照人群标本检测显,阴性38例,假阳性2例。结论PCR-探针杂交法较传统血培养法能更快速、灵敏地检测动物模型的侵袭性曲霉感染,并可用于对IA高危患者的监测。     

Detection of picogram amounts of nucleic acid by dot blot hybridization

An increasing number of human proteins isolated from cell sources are being produced for pharmaceutical use. Consequently, federal agencies have required the quantitative determination of residual nucleic acids that copurify with the potential protein products. We have conducted these assays in connection with our application for licensure of Alferon Injection. We report a sensitive dot blot hybridization assay that was used to quantitate picogram (or less) amounts of nucleic acids which copurified with human proteins isolated from recombinant (S. cerevisiae) or natural (leukocytes) sources.     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

Detection of hepatopancreatic parvovirus (HPV) in wild shrimp from India by nested polymerase chain reaction (PCR)

The prevalence of hepatopancreatic parvovirus (HPV) in wild penaeid shrimp samples from India was studied by nested polymerase chain reaction (PCR) using primers designed in our laboratory. The virus could be detected in 9 out of 119 samples by non-nested PCR. However, by nested PCR 69 out of 119 samples were positive. The PCR results were confirmed by hybridization with digoxigenin-labelled DNA probe. Shrimp species positive by non-nested PCR included Penaeus monodon, Penaeus indicus and Penaeus semisulcatus and by nested PCR Parapenaeopsis stylifera, Penaeus japonicus, Metapenaeus monoceros, M. affinis, M. elegans, M. dobsoni, M. ensis and Solenocera choprai. This is the first report on the prevalence of HPV in captured wild shrimp from India.     

Detection of gill-associated virus (GAV) by in situ hybridization during acute and chronic infections of Penaeus monodon and P. esculentus

Chronic and acute gill-associated virus (GAV) infections were examined by in situ hybridization (ISH) using a DNA probe targeting a 779 nucleotide region of the ORF1b-gene. Chronic GAV infections were observed in healthy Penaeus monodon collected from farms and healthy P. esculentus surviving experimental infection. During chronic-phase infections in both species, GAV was detected only in partitioned foci of cells with hypertrophied nuclei (spheroids) within the lymphoid organ. Acute-phase infections were observed in moribund P. monodon and P. esculentus infected experimentally with a high dose of GAV, and in moribund P. monodon collected from farms during outbreaks of disease. During acute experimental infections in P. monodon, ISH detected GAV throughout the lymphoid organ, in gills and in connective tissues throughout the cephalothorax. In moribund P. monodon collected from natural outbreaks of disease, GAV was also detected in the gills and in connective tissues of the cephalothorax, but the distribution of virus within the lymphoid organ varied. In acutely infected P. esculentus, GAV was detected in connective tissues, but was restricted to the inner stromal matrix cells and endothelial cells of intact lymphoid organ tubules. The tissue distribution of GAV identified by ISH suggests that shrimp are able to control and maintain chronic asymptomatic infection by a process involving lymphoid organ spheroids. Acute phase infections and the development of disease appear to be dose-related and involve the systemic distribution of virus in connective tissues throughout the cephalothorax.     

Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.     

Bacteriological study of shrimp, Penaeus monodon Fabricius, hatcheries in India

Shrimp hatcheries often face problems of mortality caused by diseases. To understand the bacteriological status of shrimp, Penaeus monodon Fabricius, hatcheries in India, a study of hatchery water at different points was conducted in several hatcheries located along the east and west coast of India. The species composition of the bacterial flora was also determined. The total plate counts of raw sea water on tryptic soya agar ranged from 10² to 10⁴ ml^–1, whereas it ranged from 10⁴ to 10⁶ ml^–1 in larval tanks. In the larval tanks, the proportion of Vibrio species ranged from 50% to 73%, as compared to 31% in raw sea water. A mixed bacterial flora was observed in hatchery water; however, in the larval tanks, the flora in the larvae was predominantly made up of Vibrio species. A few of the tested Vibrio isolates were non-virulent to shrimp larvae under experimental conditions. Over 90% of the strains were resistant to amoxycillin, ampicillin, cephalexin, cephazolin, cloxacillin and sulphafurazole. Most strains showed sensitivity to tetracycline, chloramphenicol, and quinolones such as norfloxacin and ciprofloxacin.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.     

Alteration of egg activation by protease inhibitors in the black tiger shrimp,Penaeus monodon

In penaeoid shrimp, contact of spawned eggs with seawater induces egg activation. However, little is known about the factors that influence egg activation in Penaeus monodon. Therefore, the main objective of the present study was to determine whether shrimp-produced proteases that are released in seawater are essential for egg activation. Female shrimp were allowed to spawn in artificial seawater containing protease inhibitors. It was shown that 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and soybean trypsin inhibitor (SBTI) inhibited egg activation. High doses of APMSF and SBTI induced only 1–2% complete egg activation. Moreover, when the APMSF- and SBTI- treated eggs were subsequently washed, egg activation did not resume. In contrast, other protease inhibitors, pepstatin A, E-64, and ethylene glycol tetraacetic acid, did not inhibit egg activation, as evident by approximately 98% complete activation. Our results suggest that serine proteases, which are most likely trypsin-like proteases, released in seawater may be involved in egg activation of P. monodon.     

Development of gene transfer technology for black tiger shrimp, Penaeus monodon

An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.