First isolation of Yersinia ruckeri from rainbow trout (Oncorhynchus mykiss) in Peru

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1 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 104 First isolation of Yersinia ruckeri from rainbow trout (Oncorhynchus mykiss) in Peru Sandra Bravo 1, Verónica Kojagura 2 1 Instituto de Acuicultura, Universidad austral de Chile. Puerto Montt, Chile. 2 Dirección Regional de Pesquerías. Departamento de Junin, Peru. Abstract Rainbow trout (Oncorhynchus mykiss) are successfully reared in hatcheries located above 3000 m altitude in Peru. Two surveys were conducted to determine the fish health status of the hatcheries in the Department of Junin, the first through a FAO project and the second by the Direccion Regional de Pesquerias. As a result of the first survey Yersinia ruckeri was isolated in nine of 17 hatcheries inspected. In the second survey, 22 of 28 hatcheries were found to be infected with Y. ruckeri. The bacteria was isolated in TSA, identified by bacteriological procedures, and the serotype confirmed using the slide agglutination reaction. Introduction Enteric redmouth disease (ERM) is an infectious disease caused by the Gram- negative bacteria Yersinia ruckeri, which is widely distributed in freshwater salmonid hatcheries. The disease has been reported from North America, Europe (Austin and Austin, 1987; Stevenson et al., 1993; Bullock, 1994) and also from Australia and New Zealand (Bullock et al., 1977; MacDiarmid, 1994; Stone et al., 1997), South Africa (Bragg & Henton, 1986), Venezuela (Alvarez et al., 1992) and from Chile infecting Atlantic salmon, Salmo salar (Bravo, 1993). Since the first isolation of the bacteria from rainbow trout in the early 1950 s in the Hagerman Valley, Idaho, USA (Ross et al, 1966), Y. ruckeri has been recognized as one of the important pathogens of rainbow trout reared in freshwater, causing a systemic bacterial disease. The apparent spread of the disease has been associated with the dissemination of fish throughout the different areas in the absence of strict control and monitoring schemes (Horne & Barnes, 1999). Farming of rainbow trout is an important economic activity in Peru. Fish are reared successfully in hatcheries located at elevations above 3000 m altitude, until they reach a weight of 250g. Freshwater is supplied from rivers, lakes and spring waters with sources at 4800 m above sea level and have the required water quality and temperature for this fish (Table 1). Like other countries, disease is one of the most important problems for commercial hatcheries in Peru, but there is little information about the diseases present in rainbow trout. Mateo in 1986 was the first to record Icthyphonus hoferi in rainbow trout by light microscopical examination of the infected tissue and histological sections (Mateo & Mateo, 1997).

2 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 105 The rainbow trout is an exotic species in Peru and was introduced for the first time from the United States in 1925 (Pedini 1984; Brenner 1994). Hatcheries are supplied with eggs from two different sources, from their own broodstock in Peru and imported eggs, mainly from USA. This paper reports on the first isolation of Y. ruckeri from rainbow trout in Peru during a survey of the fish health status of the hatcheries in the Department of Junin. Material and Methods Two surveys were carried out in the Department of Junin where there are over 40 hatcheries rearing rainbow trout. The first survey, supported by the FAO (LOA/98191/RLC), occurred from October 1-27, 1998 with 17 hatcheries inspected and the second from February 17 through to March 9, 2000 where 28 hatcheries were inspected, including nine from the first survey. Sampling at each of the hatcheries focussed mainly on fish with abnormal appearance. In the first survey 170 fish were examinated and 250 in the second survey. The behaviour and signs exhibited by the moribund and sick fish were recorded, as well as the hygiene status of the facilities and environmental parameters such as temperature, oxygen, ph and hardness (Table 1). Fig. 1: Map of Peru. Arrow shows the place where the Department of Junin is located. There was no fish disease laboratory in the area and consequently a basic laboratory was created in the regional hatchery, El Ingenio. Abnormal fish obtained from the different hatcheries were transported to this laboratory for examination of bacterial and parasitic infection. Bacteria were isolated from the kidney using TSA and BHI media incubated at February March April May June Temperature ( C) 11 C 11.8 C 12 C 13 C 13 Hardness (ppm) ph Oxygen (mg/l) Table 1. Environmental parameters of water supply for the hatchery El Ingenio during February through June, 2000.

3 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, ºC for 48 hours and identified using standard biochemical procedures (Bullock 1994; Elliot; 1994). A presumptive identification of the strain was carried out in Peru by Gram staining, oxidasa activity, motility and O/F reaction. The confirmation of Y. ruckeri was determined in Chile using the slide agglutination reaction with Type-I antisera, obtained from BiosChile. Results In addition to the fungus, Icthyophonus hoferi, identified during the first survey carried out in October 1998, plentiful colonies of Y. ruckeri were isolated from the kidney of infected fish in nine of the 17 hatcheries inspected, including one farm with net cages in a lake (Table 2). Infected fish weighing between 5 to 30 g, were lethargic, present on the water surface, and exhibited reddening around the mouth, operculum and oral cavity. Haemorrhages around the ocular cavity, and inflamed and haemorrhagic vent were also observed. The principle gross internal signs were an inflamed and haemorrhagic lower intestine, an empty gut filled with yellowish fluid, and a dark and enlarged spleen. According to the information collected from the different hatcheries, fish weighing less than 10 g showed major susceptibility to the disease and higher mortality level than the larger fish weighing more than 30 g. Outbreaks during the first survey where mainly restricted to stocks of rainbow trout fry with less than 10% accumulated mortality. In addition to these important pathogens, the opportunistic bacterium Pseudomonas sp. and a motile Aeromonas sp. were detected in fish with poor condition. Prophylactic measures and desinfection of eggs with iodophoros (Sandra: Reference to this chemical) were introduced in the country after the first survey was carried out. Outbreaks of yersiniosis were effectively controlled with oxytetracyline, the only drug available in the region, through oral treatment with 100 mg of active ingredient per kg of fish per day for 10 consecutive days (Bravo, 1998). The results from the second survey confirmed the presence of Y. ruckeri in the hatcheries located within the Department of Junin, where the bacteria was isolated in 25 of the 28 hatcheries inspected, including three farms with net cages in three different lakes (Table 2). Infected fish showed the same behaviour and Source of water 1º surve y 2º surve y Hatcheries Positive ERM Hatcherie s Positive ERM River Lake Table 2: Hatcheries positive to Yersinia ruckeri in both surveys carried out in Junin

4 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 107 signs exhibited by the infected fish recorded in the first survey. Discussion It is difficult to determine the origin of Y. ruckeri, a pathogen that is widely distributed in freshwater hatcheries in Europe, North America and also in South America (Bullock 1994; Alvarez, et al., 1992; Bravo 1993). Because these were the first fish health surveys to be conducted in Peru, it is quite difficult to determine when, or from what source, this pathogen may have been introduced. Following the first survey, prophylactic measures were introduced to avoid the introduction and spread of disease through the movement of eggs and live fish from infected to disease-free zones. However, the number of hatcheries infected with Y. ruckeri in the second survey was increased with respect to the first (Table 2). This could be explained by the movement of infected eggs and fry to uninfected areas, because Y. Ruckeri was isolated in the three main hatcheries that supply eggs and fry to the other smaller hatcheries in the first survey. Because of the characteristics of the hatcheries and the handling procedures used, it is difficult to envisage the eradication of Y. ruckeri from Peru. However, the intensity of the infection could be greatly reduced through vaccination procedures, such as those utilized in Chile after the first outbreaks of Y.ruckeri in Atlantic salmon in 1992 (Bravo, 1993). Vaccination in Chile allowed a reduction in the amount of antibacterial drugs used in the treatment of yersiniosis to near zero before a commercial vaccine became available in Acknowledgements We want to thank Ing. Luciano Chauca and people of the Dirección Regional de Pesquerías of the Department of Junin for the help given to us during the surveys. Also we want to thank to Dr. Jim Treasurer for the revision of the manuscript. References Alvarez, J.D.; B.Austin; D.A. Conroy. (1992) First outbreak of enteric redmouth in rainbow trout Oncorhynchus mykiss, Walbaum) cultured in Venezuela. Bulletin of the European Association of Fish Pathologists. 12(6): Austin, B. & Austin D.A. (1987) Bacterial fish pathogen: Diseases in Farmed and Wild Animals. Ellis Horwood, Chichester. pp Bragg, R.R., Henton, M.M. (1986) Isolation of Yersinia ruckeri from rainbow trout in South Africa. Bulletin of the European Association of Fish Pathologists. 6:5-6. Bravo S. (1993) Disease reported in pen-reared salmonids from Chile. AFS/FHS Newsletter 21(3):3. Bravo, S. (1998) Revisión de los medicamentos utilizados para el control de las enfermedades de peces en Chile. 2ª Ed. Laboratorio Veterquímica. 29 pp. Brenner T. (1994) Las pesquerías de aguas continentales frías en América Latina. Copescal Documento Ocasional N 7. Organización de las Naciones Unidas para la Agricultura y la Alimentación. FAO, Roma.

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