Detection of the etiologic agent of necrotizing hepatopancreatitis in cultured Penaeus vannamei from Texas and Peru by polymerase chain reaction

Abstract

Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease that was previously identified solely in cultured Penaeus vannamei, the Pacific white shrimp, from Texas (USA). In January 1993, a disease with similar clinical and histopathologic features to NHP was diagnosed in P vannamei cultured in Peru. Oligonucleotide primers derived from variable regions V5, V8 and V9 of the 16s ribosomal RNA gene sequence enabled polymerase chain reaction (PCR) confirmation of the diagnosis of NHP in both Texan and Peruvian shrimp. The PCR amplification products from the clinical specimens were compared to the PCR amplification product obtained from a sucrose-gradient purified sample of bacteria that was previously used to reproduce NHP. In this study, fingerprinting by restriction fragment length polymorphism (RFLP) analysis of the PCR products was used to compare the isolates. Results indicate that hepatopancreatic lesions are caused by the same bacterium in both Texas and Peru. Negative results were obtained from uninfected shrimp and shrimp infected with Vibr~o spp., as well as DNA extracted from Brucella abortus, Ehrlichia risticji, SalmoneUa enteritidis, Agrobacterium tumefaciens and Streptococcus crlae.

“Candidatus Hepatobacter penaei,” an Intracellular Pathogenic Enteric Bacterium in the Hepatopancreas of the Marine Shrimp Penaeus vannamei (Crustacea: Decapoda)

“…As had been determined previously by Loy et al (6) using the 16S rRNA gene, this bacterium is phylogenetically associated with the order Rickettsiales. Recent nucleotide sequence additions from this order of bacteria to public databases refines the placement of the 16S rRNA sequence in the genera classified as "incertae sedis," with low support branching probability (64.76%) in the family Holosporaceae (Fig.…”

“…Although the bacterium has been well characterized, in vitro attempts to culture the causative organism have remained unsuccessful (1)(2)(3)(4)(5)(6). The 16S rRNA and gyrase B gene sequences were used to elucidate the phylogenetic position of NHP-B more clearly, placing these bacteria within the order Rickettsiales.…”

Prevalencia y distribución de los principales agentes etiológicos que afectan los langostinos silvestres en Tumbes, Perú

“…Entre los patógenos bacterianos de importancia, la NHPB (bacteria de la necrosis del hepatopáncreas) produce una enfermedad severa, con mortalidades entre 20 a 90% en los sistemas de cultivo (Loy et al 1996). Esta bacteria ha sido reportada solo en peneidos de América y ha sido reconocida en diferentes especies, entre ellas: L. vannamei, L. aztecus, L. setiferus, L. stylirostris y F. californiensis.…”

Antibacterial effect of biosynthesized silver nanoparticles in Pacific white shrimp Litopenaeus vannamei (Boone) infected with necrotizing hepatopancreatitis bacterium (NHP B)

“…According to the results of the DNA quantification, the PCR reaction was performed using 1 µL of DNA if the concentration was greater than 100 ng µL -1 and 2 µL if the DNA concentration was lower than 100 ng µL -1 . One µL of the oligonucleotides described by Loy et al (1996) for diagnosis of NHP-B (pf-1: 5'-ACGTTGGAGGT TCGTCCTTCAG-3' and pr-1: 5'-TCACCCCCTTGC TTCTCATTGT-3') were added into the reaction mix. Thermal cycling conditions were as described by Loy et al (1996).…”

“…One µL of the oligonucleotides described by Loy et al (1996) for diagnosis of NHP-B (pf-1: 5'-ACGTTGGAGGT TCGTCCTTCAG-3' and pr-1: 5'-TCACCCCCTTGC TTCTCATTGT-3') were added into the reaction mix. Thermal cycling conditions were as described by Loy et al (1996).…”