Detection of Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss) fry tissues, using bacterial culture, simple PCR and nested PCR

In this study the efficacy of bacterial culture, simple PCR and nested-PCR techniques for the detection of Yersinia ruckeri in tissues of naturally and experimentally infected rainbow trout (Oncorhynchus mykiss) fry was compared. In order to detect Y. ruckeri in naturally infected rainbow trout tissues, 30 fish suspected of enteric redmouth disease were randomly collected from 6 rainbow trout farms with a history of exposure to Y. ruckeri, the causative agent of enteric redmouth disease. Also experimental challenges were performed by immersing 30 healthy fish in a bacterial suspension contained 4×108 CFU/ml Y. ruckeri. Results, from naturally and experimentally infected tissues respectively, showed that the culture method detected Y. ruckeri in 33.3% and 80% cases, simple PCR detected Y. ruckeri in 37% and 80% cases and nested PCR detected Y. ruckeri in 67% and 100% cases. Nested PCR using 2 species-specific sets of primers against the 16S ribosomal DNA gave more positive results (detection limit of 7×103 CFU/ml) and did not cross-react with other microorganisms commonly found in haemorrhagic septicemic cases in rainbow trout. Nested PCR assay could be suggested as a powerful method for early detection of Y. ruckeri from tissue samples of asymptomatic carrier fry fish. * Corresponding author’s e-mail: [email protected] Introduction Enteric redmouth disease (ERM) caused by Yersinia ruckeri, is an infectious disease in the rainbow trout farming industry that causes economic losses worldwide. The disease most commonly affects smaller rainbow trout (Oncorhynchus mykiss) and is less severe but more chronic in larger fish. Disease intensity is favored by water temperatures around 15-18°C while temperatures below 10°C suppressed the infection (Austin and Austin, 2007). Even though the disease has been diagnosed since 1981 in European countries and ERM is present in most of these countries (Furones et al., 1993), ERM has been reported from Iran only recently. Soltani et al. (1999) reported a yersiniosislike infection in farmed rainbow trout, Tehran, Iran. Infectious diseases are emerging due to imposed stress factors. Since 2002 there were several suspected ERM cases in rainbow trout from northwest and northern rainbow trout farms of the Fars province, Iran. Y. ruckeri was detected and identified by conventional biochemical and by molecular methods (Akhlaghi and Sharifi Yazdi, 2008). Phylogenetic studies of the genus Yersinia based on 16S rRNA sequencing have shown Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 178 that this genus represents a coherent tight cluster within the family enterobacteriaceae, with Y. ruckeri forming a separate group within the Yersinia five-subline cluster (Ibrahim et al., 1993). By taking advantage of the differences found in the 16S rDNA, specific oligonucleotides were designed and used in a PCR assay for detection of Y. ruckeri in tissues of inoculated and naturally infected trout (Gibello et al., 1999). In order to issue a veterinary health certificate when asymptomatic small fish are tested, only culture method is now available. This study was undertaken to compare the performances of bacterial culture and simple PCR methods already described in the literature. We also used one nested-PCR, currently developed in our laboratory for molecular diagnosis of Y. ruckeri in tissues of naturally and experimentally infected rainbow trout fry. Materials and methods Fish a) Samples from fish farms Thirty rainbow trout (1-2 g weight range) suspected of carrying Y. ruckeri were obtained from 6 rainbow trout farms situated in the northwest and west of Fars province, Iran. Suspected fish were euthanized, dissected and samples from kidney, liver and spleen were collected both for microbiological examination and molecular assay. Fish tissues were cultured aseptically by streaking a loop onto brain hearth infusion agar (BHI) plates and incubated at 25°C for 48h. Bacterial colonies were subcultured onto BHI, identified using conventional biochemical (Austin and Austin, 2007) and biotyping systems (Davies and Frerichs, 1989). Fi een milligrams of the fish tissues were also taken for genomic DNA extraction. b) Experimental infection In another experiment, 75 healthy rainbow trout (1-2 g weight range) were purchased from a recirculating system farm with no history of ERM and divided into two groups of experimental challenge (n:45) and negative control groups (n:30). A challenge protocol was designed according to Akhlaghi & Sharifi Yazdi (2008) using a bacterial suspension contained 4×108 Y. ruckeri CFU/ml (Strain YF87; FJ870985) as immersion for 30 min. Fish were kept in aerated aquaria with an average water temperature of 15°C and watched daily for two weeks. Moribund fish due to the ERM were examined and kidney, liver and spleen samples were taken as described above. Samples of kidney, liver and spleen from healthy fish (the negative control group) were also obtained aseptically for both culture onto BHI plates and PCR assays. A tissue homogenate were prepared from the fish organs a er blending with appropriate volume of saline solution. The absence of Y. ruckeri in each tissue homogenate was determined according to the method used by Gibello et al. (1999). DNA extraction from isolated bacteria The method described by Holmes and Quigley (1981) was employed for genomic DNAs extraction and molecular confirmation from bacterial colonies with some modifications. Briefly, a medium-size bacterial colony was taken from bacterial cultures and suspended in 200 μl of sterile distilled water, incubated in a boiling water bath for 15 min, Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 179 centrifuged for 5 min at 10,000 ×g and 2 μl of the supernatant was used as template for the PCR amplification. DNA extraction from tissues DNA from tissue was extracted using a commercially available kit (Qiagen DNeasy Kit, Valencia, CA, USA) according to the manufacture’s protocol. Genomic DNA was extracted from tissue homogenate (kidney, liver and spleen) of fish.