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The case of infectious hematopoietic necrosis (IHN) in rainbow trout fry – introduction of RT-PCR technique for the diagnosis of IHN virus in Croatia

Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus which causes extensive losses among salmon and trout in several areas of the world. The induced disease is typically characterized as an acute viremia with resulting hemorrhage and necrosis of major organ systems, although IHNV have been isolated from the clinical healthy fish. Cell culture based methods are used widely while in recent years molecular methods have also been identified as an acceptable diagnostic method for IHNV. In the February 2005 rainbow trout fry were delivered in our laboratory from one Croatian farm (two basins). Fry had been imported from Slovenia and accompanied by Veterinary health certificate. Nevertheless, Croatian Veterinary Inspection detected IHNV in the samples on the first day at the farm, and mortality was noticed after 18 days. Biometric data as well as detailed external and internal observations were done. Pools of different visceral organs (spleen, heart, kidney and brain) were collected and processed for cell culture propagation and RT-PCR. EPC cell lines were used for sample CPE screening and infection with IHNV strain 32/87 as positive control. We have applied two nested RT-PCR reactions, with two different primer sets, as was described previously. The RT-PCR products were analyzed by agarose gel electrophoresis. Haemorrhagic areas were detected on gills and fins were ragged. However, there were no macroscopic changes on visceral organs. Inoculated samples caused CPE on EPC cell culture. Characteristic RT-PCR products for both nested reactions were obtained (682 pb i 549 pb ; 786 pb i 323 pb). This is the first case of IHNV infection in Croatia. The presence of virus was proved using EPC cell lines inoculated with the sample material and for IHNV identification the RT-PCR technique was for the first time applied in Croatia. With this molecular method evidence of IHNV infection directly from fish tissues could be obtained in one day, with high efficiency and sensitivity.

rainbow trout fry; IHN; RT-PCR

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