Occurrence of the infectious Pancreatic necrosis virus in Rainbow trout (Oncorhynchus mykiss) farms in Hidalgo State, Mexico

During the first semester of 2002 the occurrence of the infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss) farms was investigated in the state of Hidalgo, Mexico. A total of 25 out of 29 analysed farms were positive for IPNV; however only one farm had clinically diseased fish. The spread of IPNV in this state seems to be associated with the movement of trout eggs and fry from previously infected farms. In addition, the particular form of trout farming in Mexico, reusing the water among farms, and the movement and trade of live trout of plate size (250 to 300 g), contributes to the IPNV spread. The possible route of entrance or origin of IPNV in this state was not determined. * Corresponding author’s E-mail: [email protected] Introduction The infectious pancreatic necrosis (IPN) is an acute and world-wide distributed contagious viral disease that causes high mortality in younger salmonids (Wolf, 1988; Rodriguez et al., 2003). Infectious pancreatic necrosis virus (IPNV) can persist in fish that survive the disease to become asymptomatic carriers, thereby maintaining the virus in the population by continuous elimination or transmission to susceptible lineages and other species (Wolf, 1988, McAllister & Owens, 1992). It has been established that importing and transporting fish and their products represent a risk of IPNV spread alongside with other diseases (Ahne & Negele, 1985; Wolf, 1988; Reindeer, 1999). Alternatively, mammals and birds can also act as vectors (McAllister & Owens, 1992). The clinical onset of IPN was reported for the first time in Mexico in 2000 in a group of rainbow trout fry (Oncorhynchus mykiss), originally imported as eyed eggs from North America. The virus was characterised as an IPNV Buhl strain, member of the West Buxton (A1) serotype of the aquatic Birnavirus, serogroup A (Ortega et al., 2002). Later, isolated cases of IPNV have appeared in several states of the country and although the spreading mechanisms have not yet been determined in Mexico, preliminary information seems to indicate that the different cases reported are related to the original outbreak as a consequence of fry transport (Ortega, 2003). The state of Hidalgo is one of the main producers of rainbow trout in Mexico. Fresh Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 101 water aquaculture takes place in approximately 50 farms located in 10 municipalities; however, the biggest concentration is in Agua Blanca, Mineral del Chico and Acaxochitlan municipalities with 10, 8 and 4 farms, respectively. The size and type of farms are characteristic of the Mexican trout-culture; some farms carry out only temporary fattening due to the rainfall distribution; 60% carry out intensive culture and 40% produce for self-consumption or low sale, with productions from 500kg to 60 ton per year (state government official information; unpublished data). Similar to other states of the country, Hidalgo depends on national or imported eggs and fry (Ortega et al., 2002), and the movement of live trout of commercial size (250350 g) either locally produced or from other states. Following the initial diagnosis of IPN in Mexico (Ortega et al., 2002) and the information on movement of infected fish; this study was carried out to determine the occurrence of IPNV at farm level in the state of Hidalgo, as a request from the sanitary authority. Materials and methods Study design Between the months of February and June 2002 a sampling was carried out in the 29 trout farms that stocked fish during this period, located in 10 municipalities of the state of Hidalgo. Assuming a prevalence of 10% (OIE, 2003), thirty fish of either fry size (0.5 g) or commercial size (250-300 g) were collected, depending on the fish population at the moment of sampling. The sampling was performed by first collecting the individuals with clinical signs, followed by the random collection of the remainder of the fish to complete the sampling number (Murray et al., 2003); the fish were transported alive in plastic bags with water and oxygen to Department of Aquatic Animal Health at the Centre of Research and Studies in Animal Health in the Universidad Autónoma del Estado de Mexico in Toluca, Mexico. Isolation and identification of virus Following an anesthetic overdose, samples of organs were collected for virological and histopathological studies. Small pieces of anterior kidney, spleen and pyloric caeca were collected in plastic tubes with 9 ml of Minimal Essential Medium (MEM) containing 2% inactivated bovine foetal serum, 400 UI/ml penicillin and 400 μg/ml streptomycin to aliquot 10 ml from 5-fish-pools (Murray et al., 2003). The samples were macerated and then centrifuged at 500 X g for 10 minutes at 4oC. The obtained supernatant was diluted to obtain final dilutions of 1:10 and 1:100 and 100μl were inoculated in duplicate to polystyrene 24 well plates containing monolayers of 24–hours-old Chinook salmon embryo cells (CHSE-214). The incubation was at 15°C for 1 h to allow the adsorption of the virus. Later, 1 ml of MEM containing 2% inactivated bovine foetal serum, 100 UI/ ml of penicillin and 100 μg/ml of streptomycin were added to all wells of the plate. Each plate had a well which was not inoculated serving as cellular control. The inoculated plates were observed by microscope daily for 7 days to determine the presence or absence of cytopathogenic effect (CPE). Three passages were carried out to render the sample negative (OIE, 2003). In those samples where Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 102 suspicious CPE due to the presence of IPNV was observed, a confirmation by means of indirect immunofluorescence (IFAT), specific for IPNV (Taksdal et al., 1998), was carried out. When one positive pool was found, then the sample was considered positive (Murray et al., 2003). Histological study The pyloric caeca, liver and kidney samples were fixed in 10% neutral buffered formalin, processed automatically and dyed with haematoxylin and eosin (H & E) for optical microscope examination. Epidemiological study A survey was carried out in each farm at the time of sampling, providing information about the farm, type of facilities, water origin or source. The origin of eggs or fry, exchange or transport of larger fish to and from the farm. The sanitary procedures and bio-safety measures in each farm were also considered. The collected information allowed to establish the risk of spread of IPNV, in agreement with the OIE International Sanitary Code for Aquatic Animals (2003). Results Table 1 summarizes the information of analysed farms, the localization for municipality and the risk factors considered for the occurrence of IPNV. A total of twenty-five (86.2%) out of 29 farms included in this study were positive for isolation and identification of IPNV. Besides, the virus was found in 8 out of the 10 municipalities analysed, irrespective of the number of farms in each. The IPNV was isolated in all the sampled farms from the Mineral del Chico municipality. According to the survey all these units had introduced fry from Huasca II farm. In addition, three of these farms use the same water source for culture activities and they also exchange fish of different sizes and origin. The two farms from Huasca were positive for IPNV isolation. This finding is important because they are the principal farms distributing fry in the state. Nine farms from Agua Blanca municipality were sampled and found positive for IPNV. Four farms reused water. In addition, data indicate that all the production units of this municipality use “Huasca II” as the source of fry and frequent exchange of fish of consumption size among farms and other municipalities of the same state occur. On the other hand, in the municipality of Acaxochitlan, 4 farms having independent water supply were analysed. Three of these farms were positive to IPNV and although they do not have contact among them, fish have regularly been introduced from several places in addition to “Huasca I” and “Huasca II”. The negative farm has introduced fish only from the state of Mexico. In the municipality of Tenango de Doria, the two farms analysed were positive. One of them makes regular introduction of fish of consumption size from other areas of the state and from the neighbouring state of Puebla, where no sanitary antecedents are available. The other farm acquired fish from the state of Mexico. Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 103 *First finding of IPNV in Hidalgo, related to the index case. Ortega et al. (2002) 1 Fish older than 1500 degree-days. Table 1. Relationship of rainbow trout farms studied in 10 municipalities from Hidalgo state, Mexico and their IPNV diagnoses. The probable risk factors are listed. y t i l a p i c i n u M s m r a f d e s i l a n A f o t l u s e r d n a n o i t a l o s i V N P I s r o t c a f k s i R e c r u o s r e t a W n i g i r o y r f r o / d n a s g g E f o e c n a r t n E h s i f r e d l o 1 l e d l a r e n i M o c i h C e v i t i s o p A e s u t s r i F I I a c s a u H s e Y e v i t i s o p B A y b d e s u r e t a W e t a t s o c i x e M , I I a c s a u H , n w O s e Y e v i t i s o p C B d n a A y b d e s u r e t a W I I a c s a u H s e Y e v i t i s o p D e s u t s r i F B d n a I I a c s a u H o N e v i t i s o p E D y b d e s u r e t a W e t a t s o c i x e M , I I a c s a u H s e Y e v i t i s o p F e s u t s r i F I I a c s a u H s e Y e v i t a g e n G e s u t s r i F e t a t s o c i x e M o N a c s a u H * I a c s a u H e s u t s r i F e t a t s o c i x e M , d e t r o p m I o N I I a c s a u H e s u t s r i F e t a t s o c i x e M , n w o , d e t r o p m I s e Y a c n a l B a u g A e v i t i s o p 1 e s u s r i F I I a c s a u H o N e v i t i s o p 2 1 y b d e s u r e t a W e t a t s o c i x e M , I I a c s a u H o N e v i t i s o p 3 2 d n a 1 y b d e s u r e t a W e t a t s o c i x e M , I I a c s a u H s e Y e v i t i s o p 4 e s u t s r i F e t a t s o c i x e M , I I a c s a u H s e Y e v i t i s o p 5 e s u t s r i F I I a c s a u H s e Y e v i t i s o p 6 5 y b d e s u r e t a W e t a t s o c i x e M , I I a c s a u H o N e v i t i s o p 7 e s u t s r i F I I a c s a u H o N e v i t i s o p 8 7 y b d e s u r e t a W I I a c s a u H o N e v i t i s o p 9 e s u t s r i F e t a t s o c i x e M , I I a c s a u H s e Y o c l i n o t o t A e v i t a g e n 1 e s u t s r i F e t a t s o c i x e M o N e d o g n a n e T a i r o D e v i t i s o p a e s u t s r i F I I a c s a u H s e Y e v i t i s o p b e s u t s r i F I I a c s a u H s e Y n a l t i h c o x a c A e v i t i s o p A e s u t s r i F I I a c s a u H d n a I a c s a u H o N e v i t i s o p B e s u t s r i F e t a t s o c i x e M , I I a c s a u H s e Y e v i t i s o p C e s u t s r i F I I a c s a u H s e Y e v i t a g e n D e s u t s r i F e t a t s o c i x e M o N n a l t i h c o x a c A e v i t i s o p 1 e s u t s r i F o N s e Y l a n e r a l E e v i t i s o p 1 e s u t s r i F I I a c s a u H d n a I a c s a u H s e Y o g n a l o M e v i t i s o p 1 e s u t s r i F I I a c s a u H s e Y n a p i t l a u c a Z e v i t a g e n 1 e s u t s r i F e t a t s o c i x e M o N Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 104 The only farm from the Zimapan municipality that carries out the phase of conclusion and sale of plate size trout uses fish from several zones of Hidalgo, mainly Agua Blanca. On the other hand, the positive farms from “El Arenal” and “Molango” municipalities use fry only from “Huasca II” farm. Finally, the farm from Zacualtipan municipality that obtains fish from the state of Mexico was negative; however, only 5 fish were sampled in this farm since it was close to depopulation at the time of sampling. The only farm sampled from Atotonilco, was negative. They use fry from the state of México. During the study, only one case with typical clinical signs of IPN in trout fry was observed, in addition to the viral isolation. Discussion In Mexico the presence of IPNV in rainbow trout cultures was confirmed in 2000 (Ortega et al., 2002). Since then no further studies have been conducted in Hidalgo state or the rest of the country to determine the prevalence and distribution of the virus in different rainbow trout production areas (Enríquez & Ortega, 2000). The sampling in this study was not randomized and therefore it does not represent the viral prevalence in Hidalgo. In the present study 86.2% of the sampled farms were positive to IPNV. Even though this result can be considered high, it cannot be compared with the situation of other states or the country in general, due to the absence of similar studies. In other production conditions for example in Scotland, Murray et al., (2003) estimated a prevalence of 49,6% of IPNV in salt water salmon farms and 10,6% in fresh water farms. In the study mentioned above there were different prevalence in the different sampling zones with annual variations in the period 1996 – 2001. This present study does not show prevalence per municipalities due to the small number of farms analysed and no potential seasonal variation due to the fact that only a single sampling was carried out. The spread of IPNV can be favoured by several factors (Wolf, 1988; Rodriguez et al., 2003). The epidemiological analysis shows that movement of trout fry from the Municipality of Huasca’s farms is one of the possible routes of IPNV spread in the state of Hidalgo. Most of the positive farms had some sort of contact or relationship with these farms, which are considered the main distributors of fry in the state. Huasca I carries out incubation and sale of fry imported as eyed eggs, and Huasca II carries out incubation using national or imported eggs, according to the reproductive cycle of the trout. In agreement with previous sanitary reports, “Huasca I” showed the clinical form of IPNV in 2000 (unpublished data, activities’ report of the Department of Aquatic Animal Health from the Centre of Research and Studies in Health Animal [CIESA, 2001], Universidad Autonoma del Estado de Mexico) in fry imported as eyed eggs that shared the incubation facilities with a group of eggs from the first case of IPN in the country (Ortega et al., 2002). On the other hand, “Huasca II” also showed a case of IPN by the end of 2001 in an outbreak apparently not related to the index case in Mexico and also registered in fry from imported eggs (CIESA, 2001, unpublished Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 105 data). Ahne & Negele (1985) and Wolf (1988) demonstrated that the moving of infected eggs and fry are efficient mechanisms of dispersion of IPNV in places previously known as free of infection. This can be one of the main routes of spreading in the state of Hidalgo, because during the IPN outbreaks reported in these farms, actions to control the disease were not implemented. The introduction and movement of trout eggs and fry within the state of Hidalgo and the country in general, are common practices (Zamora et al., 1999). The large deficit of this material to populate the farms forces farmers to depend on imported fish or fish from other states (Enríquez & Ortega, 2000; Ortega et al., 2002) this practise represents a risk of spreading IPNV (Bebak et al., 1998) and/or other infectious agents (Wolf, 1988). Nevertheless, the origin and time of introduction of IPNV in Hidalgo is uncertain although the suspicion exists that infected fish from the index case reported in Mexico (Ortega et al., 2002) moved to different areas of trout culture of the country including the state of Hidalgo. Due to the lack of sanitary data, there is a possibility that the virus has been present before year 2000 without showing clinical signs, or that the lack of technical experience and infrastructure necessary to make the suitable diagnosis prevented its identification (Murray et al., 2003; Rodriguez et al., 2003). It was observed in this study that in Hidalgo, like in most trout producing zones in the country, there are areas where several farms located along the rivers or small streams have to reuse the water with the associated risk of transmission of diseases since carrier animals excrete the IPNV through faeces, sexual products, urine and dead fish (McAllister & Owens, 1992; Bebak et al., 1998). McAllister & Bebak (1999) reported that IPNV can be detected in water as far as 19.3 kilometres from the outlet of contaminated farms. In addition, a high concentration of farms increases the production intensity, which is also an efficient mechanism of multiplication of the infectious agents (Rodriguez et al., 2003). In Mineral del Chico and Agua Blanca municipalities, several farms are very near to each other, which combined with the little volumes of water promote the spread and infection caused by IPNV. However, the virus was also found in farms that did not reuse each others’ water. In these cases the presence of the IPNV could be associated with the moving of eggs, fry or plate size fish (Wolf, 1988). In addition, the trade of live plate size trout (250 to 300 g) in Mexico also increases the risk of spread and transmission of IPNV and other infectious agents. In Hidalgo as well as in other areas of the country, plate size rainbow trout is transported alive or sacrificed and immediately taken to the consumption places. This particular way of trade makes it quite difficult to set up actions for prevention and control of diseases in the country (Wolf, 1988; McAllister & Owens, 1992). Despite the high percentage of IPNV isolation, only one clinical case in 0.5g fry was observed. Because many of the farms only do fattening or market-size fish production, the diagnostic analysis of this study was performed mostly in fish older than 1500 degree-days, resistant to the disease in freshwater (Ahne & Negele, 1985; Wolf, 1988) thus, most of the positive Bull. Eur. Ass. Fish Pathol., 27(3) 2007, 106 cases identified were from IPNV carrier fish (Wolf, 1988). The diagnostic process carried out in this study through the isolation in cell culture allowed the detection of any cytopathogenic viral agent and with the use of indirect inmunofluorescence the presence of IPNV was confirmed (OIE, 2003). The strain or serotype were not determined with this procedure. Due to the lack of previous sanitary data from the investigated farms, it is difficult to determine if the fish introduced from other places were carriers or if the virus had previously existed in the farms (McAllister & Owens, 1992). In the index case for Mexico, Ortega et al. (2002) confirmed the presence of the IPNV Buhl strain, member of A1 serotype from the aquatic Birnavirus, serogroup A. The finding of other IPNV strains could suggest that other different Birnavirus could be present in the system (Novoa et al., 1995). Several factors affecting the IPNV isolation could generate false negative cases. In this way, the negative result of the Zacualtipan municipality farm, where only 5 adults fish were collected, could be influenced by the sample size, since small size samples have a lower probability to have the virus (Wolf, 1988). In addition, the biggest fish without clinical disease show low viral titers, thus decreasing the probability of detecting the infection (Murray et al., 2003); the above could also be the case for negative farms. However, according to the survey these farms have less risk factors than positive farms. In this study of IPNV in Mexico, it was observed that the virus is present in most of the farms examined in the state of Hidalgo, and that there seems to be a positive association between positive farms and the trade of trout fry and live plate size fish. Finally, studies to determine the serotype and genotype of the isolated IPNV-strains, to establish their possible relationships and origin, are needed. AcknowledgementsThe authors wish to thank FUNDACIÓNHIDALGO PRODUCE A.C for the financialsupport and to Alicia López for her skillfultechnical assistance in processing samples andto Alejandro Vega Moctezuma forcoordinating the visits to the farms. ReferencesAhne W & Negele RD (1985). 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