Experimental Infection of Freshwater Crayfish (Pontastacus leptodactylus) with Infectious Pancreatic Necrosis Virus
About the author:
Rud Yu. P., Maistrenko M. I., Bezusiy O. L., Buchatskiy L. P.
Type of article:
Infectious pancreatic necrosis virus (IPNV) belongs to the family Birnaviridae and is an agent of an acute, contagious fish disease causing high mortality not only in juvenile salmonids but also in non-salmonid fishes. IPNV causes high mortalities followed by a life-long, chronic infection in the survivors. Persistently infected fish are asymptomatic that have virus in many visceral organs and can shed live virions. The mechanism of transmission and the spread of fish viruses are not fully understood, it is known that fish acutely or chronically infected with IPNV excrete the virus via faeces and sexual products. Horizontal transmission of IPNV occurs via contaminated water or cannibalism, whereas vertical transmission takes place via eggs. Atypical host fishes and bloodsucking ectoparasites, as well as other aquatic organisms and fishery equipment, must also be regarded as sources of virus infections. Nothing is known about interactions between the freshwater crayfish and viruses pathogenic for fish. Therefore, we wondered whether freshwater crayfish could play a role in the epizootiol- ogy of IPN. The reproduction of the Ukrainian isolate IPNV “Karpaty” was carried out in fish continuous cell cultures RTG- 2. Infectious titer of IPNV in RTG-2 cell line was 106,9-7,4 TCID /ml. Freshwater crayfish P. leptodactylus were 50 experimentally infected with IPNV isolate “Karpaty” by injection of 0,2 ml with infective dose of 103,0 TCID /ml. 50 Total amount of experimentally infected crayfish was fifteen. Genomic viral RNA was extracted from cell culture supernatant and crayfish organs and haemolymph using GeneJETTM RNA Purification Kit. The cDNA synthesis was conducted using RevertAidTM Premium First Strand cDNA Synthesis Kit following the manufacturer’s instructions. Then cDNA was subjected for PCR amplification. IPNV was found in crayfish organs and haemolymph up to 35 days after infection (d. a. i.). The death of crayfish was noted in period of 21-23 d. a. i. The cumulative mortality of experimentally infected freshwater crayfish was 26,6 ± 6,6 %. The crayfish excreted IPNV into the water continuously that proved by PCR assay. By the method of RT-PCR the virus infectious titer was determined comparatively in RTG-2 cell culture supernatant with the high- est virus titer and the haemolymph of crayfish during whole experiment using 10-fold dilutions assay. Our results showed that virus titer was continuously permanent in organisms of freshwater crayfish. The infective doses of IPNV in crayfish haemolymph before and after experiment did not differ impressively. Thus the results of our research indicate that freshwater crayfish could play a role in the epizootiology of IPN by means of accumulation the virus during feed the died IPNV-infected fish, and consequently favor the virus transmission. Also for demonstration of introcellular localization of IPNV and its reproduction in crayfish internal organs and haemolymph the electron mi- croscopt observation is needed and such study aspect will be preemptive in our future research. Since salmonids breeding is mainly located in the west region of Ukraine the IPNV is an economically important fish pathogen for national trout farms. The ways of transmission and potential virus sources are very burning items in disease prophilaxis, so rapid diagnostic of IPNV should be provided overall including all susceptible and non- sus- ceptible fish species, other aquatic organisms and fishery equipment. Only complete monitoring of IPNV in Ukraine has to result in total data of virus distribution in Ukraine and also to identify another strains which are widespread in Europe.
IPNV isolate “Karpaty”, experimental infection, freshwater crayfish P. leptodactylus
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Publication of the article:
«Bulletin of problems biology and medicine» Issue 4 part 1 (113), 2014 year, 70-74 pages, index UDK 576. 858