Abstract

Infectious pancreatic necrosis virus (IPNV), a significant pathogen in salmonids, can infect, but does not cause clinical disease in striped bass. Data from this study demonstrate that IPNV-infected striped bass shed sufficient virus to infect brook trout located downstream. Therefore it is critical to detect virus-carriers prior to their introduction into IPNV-free areas. Virus recovery from striped bass is increased if fish are given a steroid injection 2 weeks prior to testing for IPNV. The anterior kidney is the organ of choice for detection of IPNV in striped bass. Tissues should be kept cool, but not frozen, prior to viral assay. Storage at -70°C of IPNV-infected striped bass tissues results in almost total loss of viral infectivity within 48 hours. In contrast, tissue homogenates should be frozen not refrigerated. Detection of IPNV in striped bass will be enhanced if these modifications of diagnostic procedures are used.

Introduction

Infectious pancreatic necrosis virus (IPNV) has been long recognized to be significant pathogen in salmoid¹. Infection with IPNV does not cause clinical disease in striped bass, Morone saxatilis, but infected fish can be inapparent, IPNV-carriers². Virus-carrier trout shed IPNV in their feces³ and thus can contaminate their watershed. Recently there have been increased efforts to rear striped bass in hatcheries. Many of these facilities are located in IPNV-free areas. The goals of this study were to determine whether IPNV-carrier striped bass could infect salmonids located downstream, and to investigate methods that would increase detection of virus from IPNV-carrier striped bass.

Materials and Methods

Cell Culture and Virus

Chinook salmon embryo (CHSE-214) cells were maintained at 18°C in Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum, 2 mM l-glutamine, 200 IU/mL penicillin and 200 mg/mL of streptomycin.

The striped bass isolate of IPNV(4) was used for all studies. Passaged twice, aliquots of virus were stored at -70°C. Prior to use in experiments, aliquots of IPNV were thawed and diluted in phosphate buffered saline (PBS; pH 7.2). The internal organs from individual fish were assayed using the viral plaque assay (5).

Fish

Striped bass were maintained in 15 L tanks receiving 4 L/minute spring water (22°C except where noted) and were fed commercial trout ration. Brook trout, Salvelinius fontinalis, were maintained in 15 L tanks receiving spring water (12°C) and led-commercial trout ration. This species is one of the most susceptible to infection with IPNV'(6).

Drugs

Fish were anesthetized with tricaine methanesulfate (MS-222) for all injection and sampling procedures. The semisynthetic corticosteroid, triamcinolone acetonide (E. R. Squibb and Sons, Inc., Princeton, NJ), was injected into the peritoneum (i.p.) at 10 mg/kg.

Viral challenge of fish

Striped bass fingerlings (2- to 6-month -olds) were challenged with IPNV either by i.p. inoculation of 10(3) 10(6) plaque forming units (pfu) of IPNV or by ingestion of brook trout (1 - 3 cm length) that contained between 102 - 104 pfu of IPNV.

Twenty brook trout fingerlings (4 - 6 cm length) were placed in ranks receiving outflow from 15 4-month-old striped bass whose feces contained 10(1)- 10(4) pfu/gram. Periodically, trout were assayed for virus.

Site of virus in IPNV-infected striped bass

Tissues from IPNV-infected striped bass fingerlings were excised, weighed and assayed individually for virus. Both virus-inoculated and orally infected striped bass were used.

Recovery of IPNV from chronic IPNV-carrier striped bass injected with steroid

Striped bass yearlings that had been inoculated with IPNV 12 to 14 months previously were placed in 5 tanks (3 fish/tank). All fish were weighed and injected with steroid. One group of fish was assayed for virus at each of the indicated times (0, 3, 7, 14 and 21 days) post steroid injection.

Virus recovery after storage of IPNV-carrier striped bass

A population of IPNV-inoculated striped bass was determined to have a 95% incidence of carriers. Fish from this population group were anesthetized, placed in individual, bags (WHIRLPAC: Fisher Sci. Co.) and randomly assigned to groups that were either assayed immediately or stored at 4, -20 or -70°C prior to virus assay. After the indicated length of storage, fish were dissected and the internal organs assayed for virus.

Virus recovery after storage of homogenates of IPNV-carrier striped bass.

The tissue homogenate from individual IPNV-carrier striped bass was divided into 13 aliquots, One aliquot was assayed immediately for virus. Six aliquots were placed in whirlpac bags, six were stored in glass vials. Four aliquots (two in glass, two in plastic) from each fish homogenate were stored at each of three temperatures (4, -20 and -70°C) prior to virus assay.

Results

There was no mortality in brook trout located downstream from IPN-infected striped bass but virus (102 pfu/g) was recovered from 25% of trout tested at 4 weeks of exposure.

Virus was recovered from the anterior kidney of all IPNV-infected striped bass (Table 1). The virus was reisolated from all of the other tissues except brain.

Steroid injection of chronic IPNV-carriers increased the percent of fish from which virus could be re-isolated (Table 2), especially when the fish were tested 2 weeks after receiving the steroid.

Table 1: Tissues from which IPNV was re-isolated from infected striped bass

Table 2: Recovery of IPNV from striped bass carriers following injection with steroids

Virus was recovered most frequently from IPNV-carrier striped bass that had been stored intact at 4°C (Table 3). Virus infectivity was lost by 48 hours in IPNV-infected fish that had been stored whole at -70°C. In contrast, tissue homogenates stored at 4°C lost more of their infectivity than did homogenates stored frozen (either -20 or - 70°C) (Table 4). There was no significant difference (p <0.01, analysis of variance) between virus titers of homogenates stored in plastic bags or glass vials (Table 4).

Table 3: Recovery of IPNV from IPNV-carrier striped bass fingerlings stored prior to virus assay

Editor's Note: Table 3 has been omitted as it was not readable in the original provided pdf.

Table 4: Virus recovered stared homogenates from IPNV-carriers striped bass

Discussion

Results from this study demonstrate that brook trout can become infected with virus from IPNV-infected striped bass located upstream. Although no trout developed clinical signs of IPNV-infection and only a low percentage of brook trout became infected, it is clear that striped bass carriers can shed sufficient virus to infect susceptible fish downstream. Previous reports of water-transmitted IPNV have implicated trout as the source of IPNV infection of nonsalmonids(7). To my knowledge, this is the first account of IPNV spread from nonsalmonids to salmonid species.

Virus was reisolated from striped bass most often from the anterior kidney and spleen, occasionally from the posterior kidney and never from the brain. This pattern was seen in striped bass that were exposed to IPNV by intraperitoneal and by oral routes. The virus is frequently recovered from the anterior kidney of IPNV-carrier trout'. Our results differ from reports that suggest that the brain of trout IPNV-carriers may be the only tissue containing the virus.

Steroid injection of suspected IPNV-carrier striped bass increased the percent of virus-positive fish detected 2 weeks later. Steroids increased the detection of asymptomatic bacterial infections in trout'. In addition, previous authors have demonstrated that stress increases recovery of IPNV In infected trout(10).

The majority of studies investigating the effects of storage on IPNV recovery, have used tissue homogenates or virus-containing culture fluids but not whole fish. My data from stored IPNV-infected striped bass tissue homogenates are similar to those obtained by other authors. There was an increase of virus titer In samples stored at 4°C, therefore storage at -20 or -70°C is preferred. Various isolates of IPNV have different storage stabilities11-12 However, all samples still demonstrated some infectivity after being frozen for 2 months. The complete loss of virus infectivity in whole fish stored 2 days at -70°C was somewhat unexpected. The disadvantage of using whole fish is that one can not obtain an Initial virus titer from each sample. In the experimental group of striped bass used, there was a high ( > 95%) incidence of carriers. It is unlikely that all of the fish chosen, at random, for storage at -70°C were not virus carriers. The lack of virus infectivity presumably is a result of storage.

In summary, the demonstration of the spread of IPNV infection from striped bass to brook trout supports the recommendation that striped bass should be tested for IPNV prior to their introduction into IPNV-free watersheds (13). For virus testing purposes, I would suggest that 1) striped bass be injected with steroids 2 weeks prior to virus assay, 2) the anterior kidney and spleen be tested for virus, and 3) fish samples be kept at 4°C and assayed as quickly as possible.

References

1.  Wolf, K., Snieszko, S.F., Dunbar, C.E. and Pyle E. Virus nature of infectious pancreatic necrosis In trout. Proc. Soc. Exptl. Biol. Med. 104: 105-108 (1960).

2.  Wechsler, S.J., Schultz, C.L., McAllister, P.E., May, E.B. and Hetrick, F.M. Infectious pancreatic necrosis virus in striped bass (Morone saxatilis): Experimental infection of fry and fingerlings. Dis. Aquat. Org. (in press).

3.  Billi, J.L. and Wolf, K. Quantitative Comparison of Peritoneal Washes and feces for detecting infectious pancreatic necrosis (IPN) virus in carrier brook trout. J. Fish. Res. Bd. Can. 26: 1459-1965 (1969).

4.  Schutz, M., May, E.B., Kraeuter, J.N. and Hetrick, F.M. Isolation of infectious pancreatic necrosis virus from an epizootic occurring in cultured striped bass Morone saxatilis. J. Fish Dis. 7: 505-507 (1984).

5.  Silim, A., Elazhary, M.A.S.Y., and Lagace, A. Susceptibility of trouts of different species and origins to various isolates of infectious pancreatic necrosis virus. Can. J. Fish. Aquat. Sci. 39:1580-1589 (1982).

6.  Wechsler, S.J., McAllister, P.E, Hetrick, P.M. and Anderson D.P. Effect of exogenous corticosteroids on viremia and neutralizing antibodies in striped bass Morone saxatilis infected with infectious pancrectic necrosis virus. Vet. Immunol. Immunopath. 12:305-311 (1986).

7.  Sonstegard, R.A., McDermott, L.A. and Sonstegard, K.S. Isolation of infectious pancreatic necrosis virus from white suckers. Nature 236: 174-175 (1972).

8.  Dorson, M. Infectious pancreatic necrosis of salmonids: overview of current problems. In: Antigens of fish pathogens, D.P. Anderson, M. Dorson and P.H. Dubourget, Eds. Collection foundation marcel merieux, Lyons, France. 1982. pp. 7-32.

9.  Bullock, G.L. and Stuckey, H.M. Aeromonas salonicida: detection of asymptomatically Infected trout. Prog. Fish Cult. 37: 237-239 (1975).

10. Frantsi, C. and Savan, M. Infectious pancreatic necrosis virus: comparative frequencies of isolation from feces and organs of brook trout. J. Fish. Res. Bd. Can. 28: 1064-1065 (1971).

11. Malsberger, R.G. and Cerini, C. P. Characteristics of infectious pancreatic necrosis virus. J. Bact. 86: 1283-1287 (1963).

12. McMichael, J., Fryer, J.L. and Pilcher, K.S. and antigenic comparison of three strains of infectious pancreatic necrosis virus of salmonid fishes. Aquac. 6: 203-210 (1975).

13. Wechsler, S.J., Schultz, C.L., McAllister, P.E., May, E.B. and Hetrick, P.M. Infectious pancreatic necrosis virus in striped bass (Morone saxatilis): can be carriers of IPNV. Fish Health News 14(3):vi-viii (1985).