Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea)

Abstract

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65°C, otherwise cross-reactivity was observed with other types of bacteria. KEY WORDS: Rickettsia · Penaeus monodon · Shrimp · CrustaceaResale or republication not permitted without written consent of the publisher

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon

“…Injection of rickettsia infected Penaeus monodon homogenate into P. vannamei: The inoculum was prepared from frozen (-70°C) RLB-infected P. monodon, which originated from a shrimp farm in the southwest region of Madagascar collected in the fall of 2000 (Nunan et al 2003). A total of 15 SPF P. vannamei were injected with the inoculum and then equally distributed between the 3 aquaria.…”

“…Per os exposure of Penaeus monodon to rickettsia infected P. vannamei tissue: RLB-infected minced P. monodon tissue (Nunan et al 2003) was fed at a rate of 5% total body mass to 15 SPF P. vannamei. Approximately 4.5 g of the tissue, originating from frozen shrimp carcasses from the rickettsial disease outbreak in Madagascar, was fed to the indicator SPF shrimp at 2.5% body weight in 2 feedings daily for 2 d. The 3 replicate tanks were maintained on pelleted rations following the per os exposure.…”

“…Moribund and surviving shrimp from each of the bioassays were preserved in Davidson's fixative for histological examination (Bell & Lightner 1988) and in situ hybridization assays with a digoxigenin-labeled DNA gene probe specific to this RLB (Nunan et al 2003).…”

“…Amphipods, crabs, freshwater crayfish and prawns have been documented to be infected with RLB (Federici et al 1974, Harshbarger et al 1977, Bonami & Pappalardo 1980, Larrson 1982, Johnson 1984, Brock et al 1986, Anderson et al 1987, Krol et al 1991, Frelier et al 1992, Fryer & Lannan 1994, Bower et al 1996, Jimenez et al 1997, Edgerton & Prior 1999, Wang & Gu 2002, Nunan et al 2003. The purpose of this study was to determine whether the RLB that caused severe mortalities in Penaeus monodon could infect and cause mortalities in specific pathogen free (SPF) P. vannamei.…”

“…Cytoplasmic masses were composed of tiny bacterial rods of ~0.5 to ~1 µm that stained gram-negative with the Gram-Twort stain (Ollett 1947). The principal target tissues affected by the RLB in P. monodon included, in order of severity, the lymphoid organ, connective tissue (systemically), circulating hemocytes, fixed phagocytes, and cuticular epithelial cells (Nunan et al 2003).…”