Diagnoses, pathogeneses and epidemiologies of salmonid alphavirus diseases (SPD/SD DIAGNOSIS)

Experimental infection of Atlantic salmon by injection with the reference PD isolate induces a very mild infection with no mortality and only minor histopathological changes. Injection with new field isolates from Ireland and Norway produced in this project induces more severe disease, more like the PD outbreaks seen in the field. An experimental study by injecting Atlantic salmon pre-smolt with an Irish subtype 1 and a Norwegian subtype 3 PD virus isolate in separate tanks was performed in this project. Samples of blood and tissue from each group and from a control group of non infected fish were collected during a period of 20 weeks and examined for virus, virus RNA, antibodies, histopathology and by immune histochemistry. The results were used to follow and compare the pathological changes induced by the two different subtypes of PD virus. Results suggested that the Norwegian subtype 3 PD virus isolate may induce more severe histopathological lesions and a more prolonged infection than the Irish subtype 1 PD virus isolate, but it is recognised that there may be differences in pathogenicity among isolates from the same subtype. The sequential study has given valuable information about the pathogenesis and about the usefulness of diagnostic methods for PD. In particular, the application of sensitive real time RT-PCR testing showed that virus RNA could be detected in some fish as late as 20 weeks post infection, supporting the view that a carrier state may exist. In addition the results will be used to development a challenge model for development of vaccines against PD in Atlantic salmon.

Prior to the project approximately 15 cell culture isolates of salmonid alphavirus (SAV) had been obtained mainly by laboratories of Partners 1, 3 and 4. These had been obtained from clinical tissue specimens from outbreaks of PD and SD in Ireland, Norway and France. One of the sub-objectives of the project was to expand the number of cell culture isolates to facilitate investigation of biological (antigenic and pathogenic) and genetic diversity. This objective was achieved through efforts made by Partners 1, 3 and 4. Most of the new isolates were made with serum samples through the application of the IPX-based method for detecting viraemia in serum samples. Work with the greater range of SAV cell culture isolates has shown the existence of 3 genetic subtypes, which appear to have a geographical occurrence. In addition, investigations have shown that representative isolates display a relatively high level of antigenic similarity as determined by monoclonal antibody reactivity, and that representative PD virus isolates from Norway and Ireland exhibit similar pathogenicities in experimental infection studies. Although some work to characterise the new isolates has been undertaken, much work remains to be done and it is possible that some of the isolates made within the project may have distinctive characteristics that may make them useful commercially eg as low pathogenicity virus strains that may be suitable as live attenuated virus vaccines.

Results from epidemiological investigations conducted during the lifetime of the project suggested that there were significant differences in the susceptibility of different strains of Atlantic salmon to pancreas disease (PD). To investigate this further, an experimental trial with smolts of three different strains was performed. Three distinct strains of Atlantic salmon, (A, B and C) were allocated to 3 separate seawater tanks. Fish were challenged intraperitoneally and sampled over a 42 day period. Histological examination of pancreas, heart, skeletal muscle and brain was performed and lesions scored. Virus isolation and serology by end-point virus neutralization testing was performed on serum samples. Results at each time point were compared to identify possible strain-related differences. Significant differences were detected between smolt strains, particularly in relation to mean lesion scores in pancreas, heart and skeletal muscle. Further investigations will necessary to elucidate the mechanisms underlying these differences. These results suggest that the severity of field PD outbreaks may be influenced by the strain of fish on a given site, and that selective breeding of smolts may be one possible method of mitigating losses. As a result of this work, further field trials have been initiated by smolt producers.

Until the project started by the end of 2001, experimental Sleeping Disease (SD) had only been performed by immersion of rainbow trout fry (<10g) in SDV contaminated water. The fish size was then a limiting factor to a consistent study of the pathogenesis of SD. During the project, the experimental reproduction of SD in 1 year-old trout (100g) by intraperitoneal injection of a high concentration of SDV allowed partner 3 to get a better understanding of the development of the disease after some techniques had been developed. A full histological study performed on organs of trout sampled at regular intervals post-infection was completed and demonstrated the sequential lesions in pancreas, heart and muscle respectively. The concentration of virus in plasma, kidney and brain was optimal 7 days post-infection and then decreased to reach an undetectable level 6 weeks after infection. Neutralising antibodies were detected in the plasma of all the fish sampled 3, 6 and 10 weeks post-infection. The neutralising titre of the plasma increased within the time. During this experiment different stages of SD have been described, acute, sub-acute, chronic and recovering, which will be used by laboratories involved in fish diagnostic to evaluate the phases of the disease in affected trout farms.

The vertical transmission of SDV in rainbow trout was investigated through an experiment carried out with 20 females and 21 males originating from the virus-free facilities of Afssa-Brest. The fish were intraperitoneally injected with SDV. The virus was demonstrated to be present for a longer time in the reproductive tissues than in the sera. The nineteen lots of eggs obtained from the infected spawners were incubated separately at about 10°C. Forty eggs from each lot were sampled three times between fecundation and hatching time. Twenty of them were disinfected using Dakins solution. Both disinfected and non-disinfected eggs were analysed for the presence of SDV. The virus was evidenced in 58% of the lots before disinfection and in 47% of the disinfected lots. Moreover, typical signs of SD and high mortality were observed two months post-hatching in the progeny obtained from one of the contaminated lots of eggs. Those results demonstrate than disinfection of eyed eggs appears ineffective in preventing vertical transmission of SDV. Those results could help in evaluating zoosanitary risks associated with transfer of fertilised eggs regarding alphavirus of salmonids.

Comparative evaluation of diagnostic tests have been carried out on both experimentally- and field-derived material within the project by Partners 1, 3, 4 and 5. Following development of the viraemia and virus neutralizing (VN) antibody tests, these methodologies were transferred to other partners within the consortium. In particular, partner 4 applied these assays, albeit in a modified format. Comparative evaluation of results generated with sera obtained from sequential sampling o f Atlantic salmon experimentally infected with an Irish strain of SAV indicated a good correlation (96.7%) between results, with one partner scoring two sera positive that were found negative by the other. Evaluation of end point titres indicated that these were typically within a two-fold dilution either way. There was 100% agreement between virus isolation results on serum. Both sera and heart were also tested by (different) real time RT-PCR tests by partners 1 and 4. In both tests, sera became negative after 14 days, corresponding to the development of VN antibodies. In contrast, both assays on heart found the majority of samples up to day 35 to be positive, with individual positive results thereafter as late as day 140. The comparative evaluation also encompassed comparing the performance of different diagnostic tests. This evaluation was done where possible by using samples obtained from the same individual fish. Typically the results obtained with histology (pancreas, heart and muscle) were compared with immunohistochemistry (IHC), virology, serology and RT-PCR. Results showed that early acute infections could be diagnosed using histology, IHC (pancreas), detecting viraemia and detecting virus RNA in serum and heart tissue. Later chronic-stage infections could be diagnosed by histology, antibody detection and virus RNA detection.

A real-time RT-PCR test has been developed within the project by Partner 1. This test was developed to provide increased sensitivity for detecting SAV RNA because the conventional RT-PCR test developed by Partner 2 did not appear to have maximal sensitivity. The real time test was also developed to allow quantitative or semi-quantitative estimates of the amounts of virus RNA in experimental and clinical samples, thereby providing useful information about pathogenesis and virus pathogenicity. The primers used for the real-time test were selected following comparison of partial gene sequences of 18 SAV isolates sourced from clinical outbreaks of PD and SD in Ireland, UK, France and Norway, with conserved regions being selected to ensure maximal chance of detecting all SAVs. The test was optimised and validated using RNA extracted from dilutions of cell culture-grown virus pools and serum samples of known infectious virus titre. The developed test was capable of detecting equal to or less than 1.5 TCID 50. It was more sensitive than the cell culture growth method for detecting infectious virus in serum. In addition it was successful in detecting SAV RNA in the heart tissue samples from the majority of SPDV experimentally infected fish from 7 to 70 days post-infection.

Immunohistochemistry (IHC) is used for detection of antigens in sections made for histology. The method, as used in diagnostics of fish diseases, is usually not very sensitive. In diseased fish that suffer from infectious diseases, however, the etiologic agent is usually present in large numbers. The benefit of the IHC is that it may detect the etiologic agent in tissue lesions that are characteristic for the disease and thus it provides a fairly strong indication of a connection between the agent and the characteristic lesion(s). In the present project an infection experiment delivered tissues from PD infected and from uninfected (control) fish that were used as a basis for the development of the IHC for detection of SAV. The IHC was subsequently used on field samples, both those collected for the project and samples submitted for disease diagnostics outside the project. The method works well on pancreatic samples from acute PD-diseased fish. The IHC is thus very useful in distinguishing acute PD from acute IPN as these two different viruses give quite similar pancreatic lesions when they cause disease in salmonid fishes. This IHC did not detect SAV in PD-characteristic lesions in heart and muscle.

Initially, Partner 1 performed a retrospective study on Atlantic salmon on two sites in Ireland. The results of this indicated that virology, serology and histopathology could be used to follow the course of infection and to gain information on the duration of viraemia and the development and persistence of VN antibodies and the temporal relationship between these and histopathological changes and clinical signs. To build on these findings, three prospective studies were initiated by Partner 1 to study in detail the epidemiology of SAV infections in both trout and salmon. Trout in the first study did not become infected, but infections occurred on both other occasions. A prospective longitudinal survey for sleeping disease (SD) was carried out over a 20-week period on a caged freshwater population of farmed rainbow trout (Oncorhynchus mkyiss) in the second study. Pancreas, heart and red and white skeletal muscle were examined histologically and presence and severity of lesions recorded. Sera were tested for viraemia with Salmonid Alphavirus (SAV) and for virus neutralizing (VN) antibodies. Viraemia was detected for 4 weeks, beginning at week 6 and with a peak prevalence of 57.9% at week 7. Clinical signs and mortalities appeared at week 8. Total mortality in the study cage from week 6 onward was 6.3%, but other cages on the site had mortality levels of up to 47.2%. VN antibodies were first detected at week 9, with seroprevalence increasing to 80% at the end of the study. Geometric mean titres peaked at 1/89.4 at week 17. Histological lesions were first detected at week 7 (pancreas only), before increasing in prevalence and severity to peak at weeks 9 and 10. The majority of lesions were resolved by week 15. A prospective longitudinal study of salmonid alphavirus infection in farmed Atlantic salmon (Salmo salar L.) was initiated in post-transfer smolts on a UK farm in July 2004 and continued for 320 days in the third study. Sampling was concentrated on a single caged population (C4) with serum and tissue sampled collected and tested for viraemia, virus neutralizing (VN) antibodies, viral nucleic acid by real time RT-PCR and by histopathology. 380 sera collected between day 0 (d0) and d139 were consistently negative for both viraemia and VN antibodies. The first evidence of infection was detected at d146, when 2/20 fish were found to be viraemic and 1/20 to be antibody positive. At d153 only 1/20 fish were viraemic and one antibody positive. At the next sampling (d168) no viraemic or antibody positive fish were detected. Thereafter, single viraemic fish were detected on four occasions, including d320. The prevalence of antibody-positive fish remained low (0-5%) until d192 after which time it rose irregularly to a peak of 57.9% at d320. Real time RT-PCR was found to be more sensitive that screening for viraemia detecting a peak of 35% positive at d153 before declining. Histological lesions diagnostic for pancreas disease (PD) were observed at d146 and 153 only. Mild cardiac and to a lesser extent brain lesions were found frequently both before and after virus was detected. No clinical signs or mortalities attributable to PD occurred throughout the study. This is the first detailed report of sub-clinical infection. The results from these studies highlights the usefulness of longitudinal surveys and the techniques for detection of virus and antibodies developed within the project as diagnostic and epidemiological tools.

To better characterize this new aquatic alphavirus and to make molecular tools available, a panel of monoclonal antibodies (Mabs) directed against SDV nonstructural and structural proteins has been generated by immunizing mice with SDV-specific recombinant proteins overexpressed in E. coli as antigens. So far, Mabs against nsP1, nsP3, E2 and E1 SDV proteins have been produced and their reactivity has been characterized by Western-blot, radio-immunoprecipitation and indirect immunofluorescence assays. In addition, protein domains recognized by each Mab have been determined by immunofluorescence assay on truncated expressed SDV-derived proteins. Finally, one Mab directed against the E1 glycoprotein has been evaluated as potential tool to be used in immunohistochemistry assay on experimentally infected trout.

Prior to the project, antibodies to SAV in sera were detected using a virus neutralisation (VN) assay, which required approximately 7 days to complete, which involved relatively large reagent volumes and which required input from a person skilled in reading SAV-induced cytopathic effect. During the project Partner 1 developed a modified virus neutralisation test which could be completed in 3 days, which used a microtitre plate-based format involving very small reagent volumes, and which used immunoperoxidase (IPX) staining to detect the presence of non-neutralised virus. The immunostaining depended on the availability of SAV-specific monoclonal antibodies which were generated prior to the project and which are owned by Intervet International. The development and evaluation of the modified VN test showed that some sera contained high levels of infectious virus. This observation led to the development of a method for detecting viraemic serum samples, which is run in parallel with the modified VN test. Application of the viraemia detection assay has resulted in the isolation of many SAVs in cell culture. The application of these 2 methods involving the detection of infectious virus and virus-specific antibody has proved to be of considerable use in diagnosing SAV infections and this approach is likely to be adopted by other Partners and laboratories outside the Consortium.

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