Field and laboratory studies on Rhabdoviruses associated with Epizootic Ulcerative Syndrome (EUS) of fishes.

Epizootic Ulcerative Syndrome (EUS) is a seasonal and widely spread ulcerative disease condition of fresh and brackishwater fishes in Asia caused by a complex of etiological agents. Viral agents have been found to be associated with EUS but the role of viruses in the complex etiology has still to be identified. Further virological examinations were, therefore, conducted in this study. Two warm-water fish cell lines were established from hybrid catfish, male Clarias gariepinus x female C. macrocephalus. The HCK line was derived from head kidney and the HCT line was derived from tail tissue. Both lines were susceptible to 3 birnaviruses, sand goby virus (SGV) and infectious pancreatic necrosis virus (IPNV) serotypes Ab and Sp, 2 reoviruses, golden shiner virus (GSV) and catfish reovirus (CRV), but refractory to all 6 strains of ulcerative disease rhabdovirus (UDRV), channel catfish herpesvirus (CCV) and 1 EUS-associated reovirus (T9231). Only the HCK line was susceptible to recent EUS-associated rhabdovirus strain T9204 and tench and chub reoviruses. Using HCK, BF-2 and SSN-1 fish cell lines, 9 rhabdoviruses were successfully recovered from EUS-diseased fishes during the first 2 weeks of a 1993- 1994 epizootic. Rhabdovirus strain T9412 caused death in fry and skin damage in juvenile striped snakehead fish. A combination of this rhabdovirus and pathogenic Aphanomyces fungus appeared to induce more severe EUS disease in snakehead fish than a single infection with the fungus. EUS transmission was also experimentally achieved by co-habitation of healthy and diseased fish. Three characterised virus strains T9415, T9416 and T9429 possessed a typical bullet- or bacillus-shaped morphology and also exhibited a lyssavirus-like electrophoreotype of structural proteins similar to snakehead rhabdovirus (SHRV) and rhabdovirus strain T9204, while UDRV strains SL11, BP and 20E possessed vesiculovirus-like eletrophoreotypes. The lyssavirus-like EUS-associated rhabdoviruses, except strain T9416, were structurally and serologically similar for which the ‘serotype Sh’ is proposed while the UDRV strains are grouped as a proposed ‘serotype Ud\ Strain T9416 could not be grouped in either serotype as the homologous antiserum was capable of neutralising viruses of both serotypes. The results of this study suggest that the rhabdovirus is one of a complex of etiological agents for EUS and that at least 2 serotypes of EUS-associated rhabdoviruses are identified

Genome sequence of a novel alloherpesvirus isolated from glass catfish (Kryptopterus bicirrhis)

The 149,343-bp genome of silurid herpesvirus 1, which was isolated in Thailand from glass catfish, was sequenced. The genome was most closely related to that of ictalurid herpesvirus 2, which infects black bullhead catfish. To our knowledge, this was the first silurid catfish alloherpesvirus genome to be sequenced

Diagnostic practices for marine fish viral diseases in Thailand

The Department of Fisheries, Thailand has three institutions that are capable of virus isolation using fish cell culture system: the Aquatic Animal Health Research Institute (AAHRI), the National Institute of Coastal Aquaculture (NICA) and the Marine Shrimp Research and Development Center (MSRDC). The AAHRI is located in Bangkok while the others are in Songkhla province, south of Bangkok. Fish cell culture system was initiated in AAHRI and NICA in 1992-1993. Both institutions spent 6-12 months to develop and practice cell culture. Since then, fish cell lines have been utilized for virus isolation. Various rhabdoviruses, iridoviruses and reoviruses were isolated from diseased freshwater fishes as well as iridoviruses from cultured frog. In addition, iridoviruses and nodavirus were also isolated from diseased marine finfish. The AAHRI maintains 8 fish cell lines and 2 reptile cell lines while NICA maintains 3 fish cell lines. The MSRDC has 5 marine finfish cell lines. In the three institutions, Leibovitz -15 is the general culture medium used in both tissue culture flask and tissue culture plate systems. This medium is capable of maintaining the pH in close and open culture systems without CO2 incubation. Diagnostic practices for marine viral diseases in Thailand include virus isolation, histology and polymerase chain reaction (PCR) amplification technique. As diagnosis in virology is costly, only suspected virus-infected specimens submitted to the Aquatic Animal Disease Clinics are examined for viruses. An active surveillance program of marine viral diseases, with support from the Government of Japan-Trust Fund, has begun this year. The diagnostic procedures for marine viral diseases in the three institutions are similar to the techniques described in the Office International des Epizooties (OIE) Diagnostic Manual and Blue Book

Current status of transboundary fish diseases in Thailand: Occurrence, surveillance, research and training

The paper discussed the current status of transboundary fish diseases in Thailand. The following were given focus in the paper: status of Koi Herpesvirus in the production of common carp and koi and the status of viral diseases in the production of shrimps and prawn. Surveillance, monitoring and diagnosis of diseases of aquatic animals and the quarantine services to prevent entry of diseases of aquatic animals were also discussed

Hematology and histopathology of Coho salmon (Oncorhynchus kisutch) infected with Flexibacter psychrophilus

ThesisThesis (M.Sc.), Oregon State University, 199

Detection and identification of viral pathogens of grouper (Epinephelus malabaricus) cultured in Thailand

A virological survey was conducted in brown-spotted grouper (Epinephelus malabaricus) cultured in cages, earthen ponds and pens from April 2001 to January 2002 on the east and south coasts of Thailand. The adult grouper samples exhibited a wide range of clinical signs beginning from darkening of body color with focal distension of the skin to red boil skin, and to appearance of red spot ulcers on the body and head. Diseased fry to juvenile grouper showed darkening of body color and swirling movement. Thirty-five isolates of viruses were obtained from using freshwater fish cell lines, SSN-1 and EPC. These isolates could be grouped into two different groups of fish viruses. One viral group has been identified as a betanodavirus, which belongs to genotype RGNNV (redspotted grouper nervous necrosis virus). RT-PCR tests in this betanodavirus using specific primers to SJNNV (striped jack nervous necrosis virus) were negative, while specific primers to RGNNV showed positive RT-PCR product. The second viral group could be identified as an iridovirus. This grouper iridovirus had some level of similarities to RSIV (red sea bream iridovirus). One set of specific primers for RSIV could give positive PCR product, while the second set of specific primers gave negative PCR result. The grouper iridovirus was found different from Ranavirus. Betanodavirus could infect both adult and fry to juvenile stages of the grouper and seemed to have more significance during November to January or during the dry and cold season in Thailand. The grouper iridovirus seems to cause disease in adult size of the fish with low level of mortality and seems to infect the fish predominantly in August and September or during rainy season in Thailand. These findings indicate that two different viruses are associated with grouper and identified each as Betanodavirus and grouper iridovirus. The new isolate of grouper iridovirus has some level of variations in nucleotide sequences when compared with RSIV gene. There is a need to do more molecular study of this new isolate of grouper iridovirus as well as its pathogenesis

Detection and identification of iridoviral pathogens of freshwater fish in Thailand

An iridovirus or ranavirus survey was conducted in Thailand during April 2003 to March 2004. Five hundred and sixty-six diseased plus healthy fish and frog specimens were examined for the possibility of ranavirus infection or carrier status. The animals were obtained from fish farms, fish markets and at the Aquatic Animal Clinic of the Department of Fisheries. A total of 393 tissue extracts was prepared for virus isolation and 393 DNA extractions were prepared for ranavirus PCR detection. Only two viruses were isolated using EPC cell line. One virus isolate was obtained from diseased ghost fish, Kryptopterus bicirrhis which exhibited white muscle coloration on the dorsal part of the body and had over 50% mortality during quarantine period in a private fish farm. The other virus isolate was obtained from diseased frogs, Rana spp. The frogs were obtained from a private fish farm located in Srakaew Province, eastern Thailand. The diseased frogs were originally imported from Cambodia and showed ulcers or wounds on the rostrum and legs with over 50% mortality. The viral isolate from the ghost fish has DNA type genome and enveloped virus particles. This ghost fish virus did not belong to ranavirus because of negative result on specific ranavirus PCR detection. The viral isolate from the frogs also has DNA type genome and enveloped virus particles. This frog virus gave a positive result in PCR detection indicating, it is a ranavirus. Molecular characterization and comparison showed that this new ranavirus isolate is closely related to Rana tigrina ranavirus, ranavirus AV9803 and frog virus 3 with 99%, 98% and 98% nucleotide homology. However, based on the evolutionary tree, the ranavirus from the diseased frogs, which was originally imported from Cambodia, is a new strain or variant of the genus Ranavirus. This new strain of ranavirus is different to the previous isolates of ranavirus obtained in Thailand. Therefore, movement and control measures for frogs and fish should be in place to prevent the invasion of new ranavirus strain into Thailand and into the region

Isolation of FV3-like iridovirus from a cutaneous ulceration disease of cultured frog, Rana tigrina Cantor, in Thailand

A new frog disease occurred in frog culture farms located in Central Thailand since early 1998. The disease affects 20-100% of the frog population in affected farms. Diseased adult frogs usually exhibit ulcerative lesions on the dorsal part of the body and legs with moderate mortality (20-50%). Some frogs had red lips, ulcerated mouths and rostrums. Diseased tadpoles and small frogs appeared weak with systemic inflammation. Mortality ranged from 50-100%. Histopathological changes observed include cutaneous ulceration and systemic inflammation with exuberant hematopoiesis. No bacteria could be isolated from the kidney, spleen and liver of frogs at the early stage of the disease. Viral investigation was, therefore, conducted. Seventy virus isolates were obtained from 107 diseased frogs collected from 8 provinces using the Epithelioma papulosum cyprini (EPC) cell line at 25°C. One virus isolate (AV9803) was partially characterized. The virions were enveloped, possessed genomic DNA and hexagonal nucleocapsid morphology, and were ~128 nm in diameter. The virus completely lost infectivity when incubated at 56°C for 30 min, in organic solvent or buffer pH 3. These findings indicate that this frog virus belongs to the family Iridoviridae. DNAs of 8 virus isolates from different provinces were extracted and compared using polymerase chain reaction or PCR. Similar sized PCR products were obtained using primers that were specific to different parts of a major capsid protein gene of Ranavirus type genus FV3. Over 99% nucleotide homology was observed between one sequenced PCR product of AV9803 and the sequence of FV3. These findings suggest that a single virus species was isolated which is most likely a strain of Ranavirus. This virus strain is temporally designated as Rana tigrina ranavirus (RTRV) . The RTRV seems to be associated with cutaneous ulceration. Further infection experiments and electron micrograph examinations in the diseased frog need to be done to confirm the causative agent

Epizootic ulcerative syndrome of fishes: rhabdovirus infection and EUS induction experiments in snakehead fish

Rhabdoviral agents have long been found to be associated with Epizootic Ulcerative Syndrome (EUS) although the presence of granulomas caused by the fungus Aphanomyces invadans is currently used as the diagnostic criterium of EUS. In the past few years, rhabdoviruses have been readily isolated from EUS-infected fish during the early period of some outbreaks in Thailand, but the role of viruses in EUS remained unclear. Rhabdovirus infection and EUS induction experiments were therefore conducted in this study. The rhabdovirus strain T9412 from EUS-diseased snakehead fish was used to infect healthy snakehead. The frequency of virus re-isolation from juvenile fish infected by a number of routes decreased to 50-75% on day 7, 0-50% on day 14 and 0-25% on day 30. The virus was more virulent at 20°C than 29°C, and caused death in snakehead fry with a LD50 equal to 2.16 Log10 TCID50/ml. EUS induction in juvenile snakehead fish was experimentally achieved using both rhabdovirus and Aphanomyces invadans type species RF-6. All (20/20) juvenile snakehead developed multiple EUS lesions by day 30 at 20°C when fish were first injected intramuscularly (i/m) with virus followed by bath challenge with fungal spores. Fish which received L-15 medium by injection and were then bathed with fungal spores also developed EUS lesions although, fewer (7/20) were affected. The rhabdovirus injection alone induced only small haemorrhagic wounds at the i/m injection site in some fish held at 20(C, and most wounds healed by the end of the experiment. A similar induction experiment conducted at 29(C failed to induce the EUS disease. Results of this study indicate that the rhabdovirus is lethal to snakehead fish fry at low temperature. The virus persists for only a short period of time in the juvenile fish and induces minor skin damage. Low temperature was found to be an important factor in facilitating the host fish to succumb to rhabdovirus and A. invadans infection in the laboratory. Therefore, one possible combination of events leading to EUS in snakehead fish is low temperature and the presence of rhabdovirus and Aphanomyces pathogens