Detection And Prevalence Of Infectious Hypodermal And Haematopoietic Necrosis Virus In Penaeid Shrimp

Infectious hypodermal and hematopoietic necrosis virus (IHHNV), also called runt-deformity syndrome (RDS) is one of the major causes of diseases in cultured penaeid shrimp. It was first detected in juvenile Litopenaeus stylirostris in Hawaii. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens of other significant disease of the diseases of crustaceans listed in OIE (Office international des epizooties) manual.In this paper, diagnostic methods for the IHHNV include traditional method of histopathology, molecular methods of DNA probes and polymerase chain reaction (PCR). Simultaneous detection of IHHNV and White Spot Syndrome virus (WSSV) by multiplex PCR is also studied. Sections of histopathology showed eosinophilic intranuclear inclusions (Cowdry type A inclusions or CAIs) that are pathognomonic for IHHNV infections in connective tissues of Litopenaeus vannamei. To verify the conclusion, A pair of primers was designed from the IHHNV genomic sequence AF218266 (GenBank) that encodes for structural protein corresponding to nucleotides 2814-3516, which amplify a 703 base pair (bp) region from the virus genome. PCR amplification with the primers generated a product of the expected size from the purified IHHNV DNA of Litopenaeus vannamei and IHHNV-infected shrimp but not from the IHHNV-free shrimp, white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV). Under the optimized PCR conditions, the primers detected as little as 19.85 fg of purified IHHNV DNA, which contained only 8.83 × 103 copies of IHHNV. An effective diagnostic tool is thus provided for screening shrimp for IHHNV infections, diagnosing epidemic disease and specific pathogen free (SPF) shrimp breeding. The PCR primers for the detection of pathogen which is one of the diseases notifiable to OIE WSSV were designed from a region of the WSSV genomic sequence (GenBank AF440570). Purified WSSV DNA and nucleic acids extracted from tissue samples of clinically healthy shrimp Litopenaeus vannamei yielded an evident amplification product showing the expected mobility of a 824bp DNA fragment, thereby confirming the specificity of the primers. The sensitivity was 520fg to WSSV DNA which contained only 1.54 × 103 copies of WSSV. WSSV-free shrimp tissue DNA, IHHNV DNA and HPV DNA showed no cross-reactions.The PCR amplicon of IHHNV described above was labeled with digoxigenin (DIG)-11-dUTP as a probe used for dot blot hybridization and in situ hybridization test. It was tested by dot blothybridization for sensitivity and specificity with IHHNV DNA, DNA extracted from IHHNV-infected shrimp, DNA extracted from IHHNV-free shrimp, healthy shrimp tissues, White Spot Syndrome Virus (WSSV) DNA and Hepatopancreatic Parvo-like Virus (HPV) DNA. the sensitivity was 24.8pg to IHHNV DNA and was able to hybridize with IHHNV DNA from 26.6ng tissues DNA of IHHNV-infected Litopenaeus vannamei. No hybridization signals were observed using healthy tissue DNA, supernatant of IHHNV-free shrimp, WSSV DNA and HPV DNA. In situ hybridization, cells displayed the intense reaction with the DIG-labeled probe.The results showed high sensitivity and strong specificity of the DNA probe.The detection results above have shown evidence of IHHNV infection in the samples we collected in the study. Detection method of IHHNV and WSSV using single-round Multiplex PCR is also established. Under the optimized PCR conditions, two primer pairs amplify the target viral genome, produce two size specific amplicons of 703 and 824bp for IHHNV and WSSV respectively. The sensitivity was 2.6pg to WSSV DNA which contained only 7.70 × 103 copies of WSSV, and 99.25fg to IHHNV DNA which contained only 4.38 ×104 copies of IHHNV, 10 times lower concentration compared to those of the monoplex PCR. This assay provides a more rapid and efficient way to detect these viruses from samples in a single test.White Spot Syndrome Virus (WSSV) effected with disinfectant containing chlorine, potassium permanganate, formaldehyde, hydrogenderoxide, iquor cresoli saponatus, ethanol and tnch...