A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.     

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.     

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.     

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.     

Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR)

One possible explanation of the maintenance of many historical foci of sleeping sickness in Central Africa could be the existence of a wild animal reservoir. In this study, PCR was used to detect the different trypanosome species present in wild animal captured by hunters in the southern forest belt of Cameroon (Bipindi). Trypanosomes were also detected by a parasitological method (Quantitative buffy coat: QBC). Parasite could not be isolated in culture medium (Kit for in vitro isolation: KIVI). Specific primers of T. brucei s.l., T. congolense forest type, T. congolense savannah type, T. vivax, T. simiae and T. b. gambiense group 1 were used to identify parasites in the blood of 164 animals belonging to 24 different species including ungulates, rodents, pangolins, carnivores, reptiles and primates. Of the 24 studied species, eight were carrying T. b. gambiense group 1. Those parasites pathogenic to man were found in monkeys (Cercocebus torquatus and Cercopithecus nictitans), in ungulates (Cephalophus dorsalis and C. monticola), in carnivores (Nandinia binotata and Genetta servalina) and in rodents (Cricetomys gambianus and Atherurus africanus). 13 species (54%) were carrying T. brucei s.l. identified as non-gambiense group 1.     

Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay

Clonorchis sinensis is a fish-borne trematode endemic to East Asia, which infects over 35 million people globally. In the study described here, we developed a nested polymerase chain reaction (PCR) method for the specific and reliable detection of C. sinensis. The primers designed from the nucleotide sequence data derived in this study were evaluated for their specificity and sensitivity for the detection of C. sinensis. The specific amplification products were obtained only with C. sinensis and no amplifications occurred with the DNA of closely related trematodes including Opisthorchis viverrini demonstrating the specificity of the assay. The novel PCR method described here will be useful for the quarantine of fishery products and evaluation of transmission status of clonorchiasis in the endemic areas.     

Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization,dot blot hybridization and PCR amplification

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.     

Antemortem detection of mouse parvovirus and mice minute virus by polymerase chain reaction (PCR) of faecal samples

Parvoviruses remain one of the most common viral infections seen in laboratory mouse colonies. The purpose of this study was to develop an antemortem polymerase chain reaction (PCR) assay to detect mice infected with mouse parvovirus-1 (MPV) and mice minute virus (MMV) using faecal samples. The MMV PCR assay consistently detected as few as 100 plasmid copies of MMV in faecal samples, while the MPV PCR assay detected as few as 10 plasmid copies of MPV. Faecal pellets from infected mice held at room temperature from 1 to 7 days tested positive by MMV and MPV PCR, respectively. This demonstrates that parvovirus DNA is stable in faecal samples kept at room temperature. PCR assays were also used to follow the length of MMV and MPV shedding in faeces from SENCAR mice, which were endemically infected with multiple agents. MMV faecal shedding was detected in 60-70% of the mice 5-7 weeks old, and by 13 weeks of age, faecal samples from all mice were negative for MMV. MPV faecal shedding was detected in 90-100% of the mice 5-11 weeks old; however, by 19 weeks of age, faecal samples from all mice were negative for MPV. These findings confirm that faecal shedding occurs for a limited time and suggest that 5-9-week-old mice are the most appropriate age group in endemically infected mice for faecal testing by MMV and MPV PCR.     

Rapid sexing of murine preimplantation embryos using a nested,multiplex polymerase chain reaction (PCR)

The objective of this study was to develop a rapid and efficient means of sexing murine preimplantation embryos at the 4- to 8-cell stage of development. To achieve this goal, a nested, multiplex polymerase chain reaction (PCR) was optimized using DNA from male and female mice and primers specific for X- (DXNds3)- and Y- (Sry,Zfy) gene sequences. Sensitivity of the assay was measured using groups of 4, 2, or 1 blastomere from dissociated embryos. Efficiency was evaluated using single blastomeres obtained by embryo biopsy. Accuracy of sexing was determined by comparing single-cell results with those of matched blastocysts. Robust amplification of male (XY) and female (XX) gene sequences was obtained in less than 6 hours. The percentage of male (3 bands) and female (1 band) reactions for groups of 4, 2, or 1 blastomere was 100% (6/6), 100% (15/15), and 94.4% (17/18), respectively. Assay efficiency for single, biopsied blastomeres from 4 to 8 cell embryos was 95.8% (207/216). For male and female embryos, sexing of single blastomeres accurately predicted results of matched blastocysts, 100% (10/10) and 100% (13/13), respectively. Simultaneous amplification of one X- and two Y-gene sequences ensured correct interpretation of sexing reactions. Short thermal cycling times and minimal tube handling increased the assay speed and decreased the potential risk of contamination. Mol. Reprod. Dev. 49:261–267, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Detection of spinal muscular atrophy carriers by nested polymerase chain reaction of single sperm cells

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a carrier frequency of approximately 1 in 40. Approximately 95% of patients have homozygous deletions of exon 7 and/or 8 of the SMN1 gene. Carrier testing for SMA is relatively complex and requires quantitative polymerase chain reaction (PCR) of genomic DNA to determine SMN1 copy number. The purpose of this study was to assess the feasibility of carrier testing for SMA in males, by nested PCR analysis of SMN1 deletions in single sperm cells. A nested PCR method was developed to amplify SMN1 exon 7 in single cells. Restriction enzyme digestion with DraI was used to differentiate between the highly homologous SMN1 and SMN2 genes. Single sperm cells from five known SMA carriers and six noncarriers were analyzed. Among the five carriers, a total of 132 single sperm cells were analyzed and SMN1 exon 7 deletion was detected in 68 cells (51.5%). In contrast, among the six noncarriers, a total of 136 single sperm cells were analyzed. Of these, an apparent SMN1 exon 7 deletion was detected in four sperm cells. This was interpreted as an allele dropout (ADO) rate of 2.9%. We conclude that nested PCR of SMN1 exon 7 is an accurate and reproducible method for detection of SMA male carriers with a SMN1 deletion.     

Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa . A total of 347 leafhoppers representing 16 species were captured and sampled from American elm ( Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta , regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa . Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.     

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.