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Risk Factors For Transmission Of Infectious Salmon Anaemia Virus (ISAV) Between Atlantic Salmon Farms In Chile: Preliminary results Arriagada G (1), Castillo F (1), Yatabe T (1), Cristi R (2), Imilán M (2), Mancilla J (2), Urcelay S (1) (1) Faculty of Veterinary and Animal Sciences, University of Chile, Santiago, Chile. (2) Marine Harvest Chile, Puerto Montt, Chile. Contact: [email protected] ABSTRACT In mid 2007 infectious salmon anaemia virus (ISAV) was detected and isolated from farmed Atlantic salmon in Chile. Infected fish showed typical signs of the disease, being the first time that ISA was reported as its clinical form in the southern hemisphere. ISAV spread out through southern Chile causing severe economic and social impacts. The objective of this study was to identify key factors associated with the spread of ISAv between Atlantic salmon farms in the Chilean epidemic. Variables significantly associated with a shorter time to infection were bath treatments against sea lice, proximity to confirmed positive fish farms, marine sites visited by vessels coming from quarantine zone, and high level of bird predation. Results suggest that ISAV was disseminated locally through sea water, in addition to mechanical vectors (fomite) such as transport boats, responsible for large-scale spread. Birds and shared equipment could have played a significant role in transmission between fish farms. KEYWORDS Infectious Salmon Anaemia, ISAV, Chile, Transmission, Risk Factor, Atlantic salmon INTRODUCTION Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon (Salmo salar L.) caused by the orthomyxovirus infectious salmon anaemia virus (ISAV). Because of its infectiousness and economic impact ISA has been listed as a notifiable disease by the World Organisation for Animal Health (OIE). ISA outbreaks were first reported in Norway in the mid-1980s, since then the disease has been informed in Canada (1996), Scotland and Shetland Islands (1998), the Faroe Islands (2000) and the United States (2001). ISAV was isolated in Chile from coho salmon in 1999, but associated with atypical signs (Kibenge et al., 2001). In July 2007, an ISA outbreak was reported for first time in farmed Atlantic salmon from Chile, with increased mortality and consistent clinical signs. Later, ISAV was confirmed by PCR and cell culture. After one year since the beginning of epidemic, ISAV is prevalent in most of the growing areas of X and XI regions. Economic impacts until 2008 were estimated in more than US$ 15 million. It is believed that transmission of ISAV occurs by passive transmission via sea water, transmission via escaped infected salmonids or wild salmonids, and transmission via vessels between marine sites, included wellboats (Nylund et al., 2007). Epidemiological studies have indicated that ISAV can be transmitted through sea water within a radius of 5 km approximately, being proximity to an outbreak fish farm or fish processing plants a risk factor (McClure et al., 2005). Large scale spread could be explained by live fish movement and transport vessels such as wellboats (Murray, 2002). Among some others risk factors described for ISAV positive diagnosis at fish farm level are visits of vessels, sharing of equipment between sites, proximity to ISA positive or in outbreak fish farm and to processing plants, lack of farm level biosecurity measures, absence of fallow period, presence and burdens of sea lice, general health status of fish, adaptability of smolts to marine environment, stocking density and initial number of fish stocked. The objective of this study is to identify determinants for transmission of ISAV between Atlantic salmon fish farms in the Chilean 2007 ISA epidemics. METHODS Study unit, study population and study time period Study unit was set as the production cycle, determined by the smolt stocking and the harvest or culling. A total of 54 production cycles were recruited from Marine Harvest Chile (MHC) Atlantic salmon farms in operation by June 2007 (study population). In some cases, were included two production cycles from one fish farm. Study time period was from June 2007 to September 2008. Case definition A case was defined as any production cycle with at least two ISA positive diagnoses by real time reverse transcription-polymerase chain reaction test (RT-PCR) during its operation. Diagnostics were obtained from a laboratory network certified by the National Fisheries Service (Sernapesca). Variables selection and data collection Variables related to the entry of the virus into the fish farm, as well as, those associated with host susceptibility, were taken into account. Source of information was scientific literature dealing with ISAV transmission risk factors (cited above), and empirical experience of the veterinary team of MHC. Data was collected from a variety of sources. Main source was the productive information system of MHC Superior™, in addition to a questionnaire applied to the veterinary team that was complemented with several technical meetings and interviews. Most variables were recorded objectively, but others were based on the veterinary team opinion. Statistical procedures Production cycles, as the study units, have variable time duration, therefore the time-at-risk of each one are different, making them non-comparable with respect to the risk for acquiring the virus. Thus, survival analysis was choosen as the statistical tool for identify risk factors, setting the failure event to whether or not the fish farm was positive for ISAV during a particular production cycle, so the response variable was the time (in weeks) between the smolt stocking and the first ISAV positive result by RT-PCR, whenever it was later confirmed with a second RT-PCR positive result. Descriptive analysis was carried out to explore predictor variables with respect to the response variable. Univariate analysis was subsequently performed in order to determine significant associations between the predictor variables and time to first positive result. Log-rank test and Kaplan-Meier technique were used to compare survival functions for categorical predictor variables. Continous predictor variables were tested by univariate Cox regression. Variables that presented unconditional association (P < 0.25) in the univariate analysis were applied in a multivariate Cox regression model. Final model was built using backward elimination method, removing least-significant variables by the Wald statistic (P < 0.05). Statistical analyses were carried out with SPSS 13 (SPSS Inc.). RESULTS Fifty-four production cycles were included in the analysis, of which forty met the case definition. Twenty-nine variables were analyzed, and eleven showed unconditional association (P < 0.25) with the response variable. Only four significant variables (Wald-test P < 0.05) were kept in final model: (1) bath treatments against sea lice (Caligus rogercresseyi), (2) presence of ISA positive fish farms in a radius of 10 km, (3) bird predation level and (4) visits of vessels coming from quarantine zone (Table 1). Table 1. Cox regression model for risk factors for the time to first ISAV positive result in MHC Atlantic salmon farms during 2007. Category Hazard ratio Bath treatments against sea lice Yes No 7.7 − < 0.001 Presence of ISA positive fish farms in a radius of 10 km Yes No 2.7 − 0.008 Low Medium High (ref.) 1.2 5.4 Yes No 2.5 − Variable Bird predation level Visits of vessels coming from quarantine zone P -value 0.801 0.011 0.043 DISCUSSION Significant risk factors Bath treatment against sea lice was the variable that showed the strongest association with the response variable, being a significant risk factor for the time to the first ISAV positive result. Until 2007, the oral treatment with emamectin benzoate (SLICE™) was the only drug allowed by the Chilean sanitary authority, year when baths with hydrogen peroxide and with deltamethrin were authorized. Because of a suspected development of resistance to emamectina benzoate and the high burdens of sea lice infestation, baths with deltamethrin were extensively used as sea lice control method during 2007. As is known, bath treatment is a stressful method which could increase the host susceptibility to infection. On the other hand, there is a suspicion about the role of C. rogercresseyi in transmission of ISAV in Chile (Hamilton-West et al., 2008), so the fact of treat with deltamethrin may reflects a high burden of sea lice in a particular fish farm, which could be the responsible of carry on ISAV inside the fish farm along with the effect of currents. Finally, sharing of equipment and personnel for baths may have helped to spread ISAV between MHC fish farms. Several cut-off points were set to categorize proximity to ISAV positive fish farms (radius of 2, 5, 10 and 15 km); each one of them was tested in the univariate analysis. 10 km cutoff point represented the largest radius that that was associated to a significant hazard ratio greater than 1, thus it was included in the Cox regression model. This result suggests that, in Chilean ISA epidemic, ISAV could be transmitted through sea water in a wider range than those proposed by other epidemiological studies that have set this action range in 5 km (McClure et al., 2005). This wider action range may be explained by the dominance of high daily fluctuations of tides (> 6.5 m) and associated strong tidal currents in the Chiloé inner sea, which are additionly affected by the complex topography caracterized by narrow bays and channels and changes in bathymetry. The cumulative percentage of mortality attributed to bird predation was categorized in three levels: (1) ≤ 0.1%; (2) > 0.1% to ≤ 0.3%; and (3) > 0.3%. Only the high bird predation level was significantly associated with a shorter time to the first positive result, in comparission with the low bird predation level (reference category). This result suggests that birds which visit the fish farms in search of food may act as mechanical vectors of the ISAV, spreading the virus between different marine sites, as noted by Murray (2002) who signed birds as natural vectors or ISAV. Another possibility is that bird predation could play a role increasing fish susceptibility to ISAV infection, hypothesis suggested by Gustafson et al. (2007). The main kind of birds that are sighted in Chilean fish farms belongs to the Phalacrocoracidae (cormorants) and Ardeidae (herons and guairabos) families, all of which are piscivorous. Seagulls and vultures (jote) also visit marine sites to feed on dead fish (J. Jiménez pers. comm.). Fish farms that were visited by boats coming from quarantine zones during its operation showed a significant shorter time to the first positive result wtih respect to those marine sites that were visited only by ships coming from free or surveillance zones. These results suggests that transport boats could have played a role in ISAV transmission in the Chilean epidemic, as noted by Murray (2002) and by McClure et al. (2005), for the case of Scotland and New Brunswick, respectively. Some variables described as risk factors for ISAV transmission by other studies such as visits by well-boats (Murray, 2002) or fish stocking density were not significant, possibly due to: (1) lack of variability associated with common management schemes determined by the single company that participated in this study, and/or (2) relatively small number of production cycles included in this study. CONCLUSIONS It is possible to conclude that −for the ISA epidemic in Chile− ISAV could be spreaded locally through the sea water within a radius of 10 km, whilst transport vessels could be responsable for large-scale transmission. Birds and shared equipment and personnel could have played a role in the ISAV transmission as mechanical vectors. Stressful management methods such as deltamethrin baths may have increased the susceptibility of fish to infection. Further efforts must be done to include a larger number of fish farms from MHC and other salmon farming companies in order to confirm the indetified risk factors, and to test additional possible risk factors. REFERENCES – Gustafson, L.; Ellis, S.; Robinson, T.; Marenghi, F.; Merrill, P.; Hawkins, L.; Giray, C. and Wagner, B. (2007) Spatial and non-spatial risk factors associated with cage-level distribution of infectious salmon anaemia at three Atlantic salmon, Salmo salar L., farms in Maine, USA J. Fish Dis. 2007, 30, 101–109. – Hamilton-West, C.; Arriagada, G.; Lara, M.; Valdés, P.; Gallardo, A. and Urcelay, S. (2008). Epidemiological description of Sea Lice (Caligus rogercresseyi) situation in southern Chile in August 2007. J. Fish Dis. (In press). – Kibenge, F.S.B.; Gárate, O.N.; Johnson, G.; Arriagada, R.; Kibenge, M.J.T.; Wadowska, D. (2001) Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile. Dis Aquat Org. Vol. 45: 9–18, 2001. – McClure, C.A.; Hammell, K.L. and Dohoo, I.R. (2005) Risk factors for outbreaks of infectious salmon anemia in farmed Atlantic salmon, Salmo salar. Prev. Vet. Med.72 (2005) 263–280. – Murray, A.G. (2002) The epidemiology of infectious salmon anemia in Scotland. International response to infectious salmon anemia: prevention, control, and eradication: proceedings of a symposium; 3–4 September 2002; New Orleans, LA. Tech. Bull. 1902. Washington, DC. – Nylund, A.; Plarre, H.; Karlsen, M.; Fridell, F.; Ottem, K.F.; Bratland, A.; and Sæther, P.A. (2007) Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar). Arch Virol (2007) 152: 151–179.