Detection of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon using PCR-ELISA.

A rapid and sensitive PCR-ELISA has been developed for detection of hepatopancreatic parvovirus (HPV) in Penaeus monodon. The specific primer set amplified 156 bp fragment and could detect as a little as 0.01 fg of purified HPV DNA which equivalent to three viral particles. No cross-reactivity was observed when nucleic acid templates from white spot syndrome virus, yellow-head virus, monodon baculovirus and shrimp were tested. The crude DNA simple prepared from hepatopancreas can be used as DNA template and provide a favorable result. Using this technique for detection of HPV infection in 87 carrier shrimps revealed the higher sensitivity and efficiency of detection when compared to histological examination and conventional PCR. Sixty-two percent infection was detected by PCR-ELISA from samples with HPV negative diagnosed by histological examination. Therefore, this sensitive and specific method is promisingly useful for early detection of HPV infection in broodstock, carriers and for ex situ application where large numbers of samples can be analyzed simultaneously.

PCR based method for identification of zoonostic Brugia malayi microfilariae in domestic cats.

The survey of 326 human blood samples in the endemic area of Surat Thani and Narathiwat, the provinces in the south of Thailand, revealed that 5 of them were infected with Brugia malayi. Similarly, 53 feline blood samples were also investigated and found that 15 of the domestic cats were also infected with B. malayi. Upon the examination of human and feline blood specimens, a pair of human and domestic cat stayed in the same house and region. The periodicities of human B. malayi and feline B. malayi were similar as well as the results of Giemsa and acid phosphatase stained blood films of microfilaria positive cases. Likewise, the PCR-RFLP profile of Hha I repeat genes and PCR amplification of Trans-Spliced Leader Exon I (SLX) demonstrated that 15 samples the feline B. malayi were the same as those of human B. malayi. The data indicated that domestic cat plays an important role as the animal reservoir for B. malayi in the endemic areas of Thailand.

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.

Detection of Plasmodium falciparum and Wuchereria bancrofti infected blood samples using multiplex PCR.

A rapid and sensitive multiplex PCR has been developed for the diagnosis of multiple parasitic infection in human blood. Infection is detected by a single multiplex PCR reaction containing two pairs of oligonucleotide primers whereby each primer is specific for each parasite species. These primer sets amplified 400 and 450-bp fragments for Wuchereria bancrofti and 208-bp fragment for Plasmodium falciparum. The PCR products derived from each parasite species were visualized in ethidium bromide-stained agarose gels, therefore allowing the rapid identification of any, or all, of the two human parasites, if present, in a single amplification reaction. This multiplex PCR was very sensitive with the ability to detect the presence of as little as 10 pg of parasite DNA. The primers used in this multiplex PCR also showed highly specific amplification of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to analyse 36 human blood samples of Myanmar workers in the endemic area at Tak Province, Thailand. Two samples showed the multiple infection, 27 samples were either infected with W. bancrofti or P. falciparum and seven samples were negative for both methods. The high sensitivity, specificity and rapidity of this multiplex PCR method make it suitable for large-scale epidemiological studies and following of drug treatment.

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.