- Email: [email protected]
Effects of infectious pancreatic necrosis virus (IPNV) on appetite and growth in Atlantic salmon, Salmo salar L.
Aquaculture 163 Ž1998. 185–193
Effects of infectious pancreatic necrosis virus ž IPNV/ on appetite and growth in Atlantic salmon, Salmo salar L. B. Damsgard ˚ ) , A. Mortensen, A.-I. Sommer Norwegian Institute of Fisheries and Aquaculture, 9005 Tromsø, Norway Accepted 28 February 1998
Abstract The effects of infectious pancreatic necrosis virus ŽIPNV. infection on appetite and growth in Atlantic salmon, Salmo salar L., were studied under experimental conditions. Eight groups of 60 fish Žage 1 q , initial weight 172 g. were bath infected with IPNV, while two groups served as controls. Virus titres were quantified in kidney and pylorus, both prior to the experiment and eight times from 9 to 44 days after infection. Feed intake of individual fish was measured using X-radiography at sixteen occasions from 7 to 44 days after infection. During the experiment, no mortality occurred in the infected or in the control groups. In infected fish, the virus titre in both kidney and pylorus increased significantly until 16 days after infection, to approximately 10 6 –10 7 p.f.u. gy1 tissue, while no virus was detected in the control groups. Between 16 and 44 days after infection, the virus titre in pylorus decreased significantly to a level of 10 3 –10 4 p.f.u. gy1. From approximately 20 days after infection, feed intake and specific growth rates were significantly lower in infected fish compared to uninfected fish. Few fish completely lost their appetite, and the study revealed that IPNV-infected fish may have relative high virus titres before any changes in appetite or growth could be detected. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Virus; IPNV; Appetite; Feeding; X-ray; Salmo salar
1. Introduction Many animal diseases and infections are accompanied by a loss of appetite ŽHart, 1988., and feed intake and feeding behaviour are thus generally used to indicate the )
Corresponding author. Tel.: q47-77-62-91-60; fax: q47-77-62-91-80; e-mail: [email protected].
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 4 2 - 7
186
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
health status of fish. Feed intake is also one of the most important factors determining the economic outcome of fish farming. Thus, information of changes in appetite is essential to ensure optimal growth and keep feed wastage to a minimum. Infectious pancreatic necrosis virus ŽIPNV. is an aquatic birnavirus ŽDobos and Roberts, 1983.. The virus causes acute infections, often with fatal outcome, primarily in juvenile salmonids ŽSnieszko et al., 1959; Hill, 1982; Wolf, 1988.. The incubation time of IPNV is approximately 2 to 4 weeks, and the mortality may range from zero to 100% in juvenile fish, depending on the serotype encountered, degree of infection, fish age, species, and water temperature Žreviewed by Wolf, 1988.. Several IPN outbreaks in Atlantic salmon, Salmo salar L., with high mortality during smolting and after transfer to sea cages have also been observed ŽKrogsrud et al., 1989; Smail et al., 1992; Smail et al., 1995; Jarp et al., 1995.. The risk that Atlantic salmon develop IPNV in sea water is affected by origin of the smolts, handling stress and geographical locations of the fish farm ŽJarp et al., 1995.. As in other salmonids, Atlantic salmon surviving an outbreak of IPNV may become life-long asymptomatic carriers of the virus ŽSwanson and Gillespie, 1979; Smail and Munro, 1985.. Despite a general observation of appetite loss in IPNV-infected fish, no experimental studies have monitored individual feed intake and growth of infected fish. To evaluate if loss of appetite or growth reductions can be associated with an IPNV infection in Atlantic salmon, we studied the effects of IPNV infection on appetite and growth in post-smolts, using X-radiography to monitor individual feed intake during six weeks after infection.
2. Materials and methods Ten groups of hatchery reared Atlantic salmon Ž1 q , initial weight 172 g, n s 60 in each group. were reared in 500 l tanks in the Fish Health Laboratory at the Aquaculture Research Station in Tromsø, northern Norway Ž708N 198E., from 3 October 1995 onwards. The fish were individually tagged ŽFTF-69, Floy Tag and Manufacturing, Seattle, USA.. Water temperature was maintained at 108C, salinity was held at 10‰, and the photoperiod simulated to match the natural photoperiod in Tromsø. The fish were fed commercial dry feed pellets ŽFelleskjøpet, Sandnes, Norway; FK-Vekst, 4 mm pellets. in excess, using automatic disc feeders. Three weeks after tagging Ž23 October., eight of the groups were infected with IPNV, while two groups were left uninfected, but were otherwise treated identically. The IPNV used for infection was isolated from Atlantic salmon juveniles after a natural outbreak and passage three times in chinook salmon embryo ŽCHSE-214. cells ŽFlow, Herts, England; ATCC no. CRL 1681.. The stock virus preparation contained 2 = 10 8 TCID50 mly1 Žtissue culture infective doses at 50% endpoint., and was stored at y808C. Experimentally IPNV infection was performed by keeping the fish for 4 h at 108C and 10‰ salinity in 100 l oxygen-saturated water, containing about 2 = 10 5 TCID50 mly1 IPNV. At eight times during the experiment Žday 9, 14, 18, 23, 30, 35, 39 and 44 after
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
187
infection., we sampled head kidney and pylorus from 10 fish from each group. The organs were weighed and homogenised into a 10% Žwrv. suspension in minimum essential medium ŽMEM, Gibco, Renfrewshire, Scotland.. The pylorus homogenate was initially centrifuged for 10 min at 2000 = g. The pylorus supernatant and the kidney homogenate were clarified for 15 min at 12 000 rpm in an Eppendorf centrifuge and the supernatants were used for virus titre assay. CHSE-214 were grown at 208C in MEM, supplemented with 1% Žwrv. non-essential amino acids, 1% L-glutamine and antibiotics. For cell growth purposes 8% foetal calf serum ŽFCS, Gibco. was added to the medium, and 2% FCS was used when the cells were virus infected. For virus titre assays, the cells were seeded into 96-well cell culture plates ŽCostar, Cambridge, USA.. Ten-fold dilution of samples were added on to CHSE-214 cells and observed for cytopathic effect in the monolayers after seven days of incubation. TCID50 were calculated according to a method of Reed and Muench, and one TCID50 dose corresponds to 0.70 plaque-forming units Žp.f.u.. ŽDulbecco, 1980.. The minimum detectable titre was 25 p.f.u. gy1 of tissue Žkidney, pylorus.. The prevalence of IPNV was expressed as the frequency of fish with detectable virus. The IPNV carrier condition was investigated by use of kidney homogenates from 20 fish sampled prior to the experimental IPNV infection and in uninfected fish from the control groups throughout the study. The fish had no known history of IPN, and none of these samples showed cytopathic effect after two additional passages on to new CHSE-214 cells. At regular intervals after infection Ždays 7, 9, 11, 14, 16, 18, 21, 23, 28, 30, 32, 35, 37, 39, 42 and 44., we measured feed intake of individual fish using X-radiography ŽTalbot and Higgins, 1983; Jørgensen and Jobling, 1989; Jobling et al., 1995.. The control groups were measured at four occasions during the experiment Ždays 9, 23, 30 and 44.. Each group was randomly measured two times during the experiment. During the X-ray sessions, feed was replaced with pellets of identical nutrient composition and physical qualities, but formulated to contain 1.5% by weight of X-ray dense glass beads Žsize 8.5 Ballotini, Jencons, Leighton Buzzard, Bedfordshire, England.. Fish were anaesthetised using benzocaine, weighed to the nearest 0.1 g, and X-ray photographed ŽTodd Research machine, 3 s exposure, 80 kV, AGFA Structurix D7 film.. Glass beads in the stomach and intestine of the fish were counted, and feed intake in grams were calculated using a calibration curve describing the relationship between numbers of glass beads Ž B . and the weights of feed Ž F; g.: F s 0.013 = B y 0.061 Ž r s 0.99, n s 19, P - 0.001.. Weight-specific feed intake ŽFI. were expressed as mg feed per g fish per day Žmg gy1 dayy1 .. Specific growth rates ŽSGR; % Žb.w. dayy1 .. between two measurements were calculated as: SGR s 100 = Žln W2 y ln W1.rt, where W1 is the weight at time 1, W2 is the weight at time 2, and t is the time in days between measurements. Data are presented as means " standard error ŽS.E... Non-parametric Mann–Whitney U-tests were used to examine for differences in virus titres, while t-tests were used to examine for differences in weight-specific feed intake and specific growth rates. A probability level of less than 0.05 was considered as significant.
188
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
3. Results Prior to the experiment, there were no differences in body weight between the 10 groups, and no virus was detected Žvirus titre - 25 p.f.u. gy1 .. In the control groups, no virus was detected during the experiment Ž9, 23, 30 and 44 days after infection.. In infected fish, the virus titre in both kidney and pylorus increased 2–3 weeks after infection, and thereafter decreased until the end of the experiment ŽTable 1, Fig. 1.. The increase in virus titre between day 9 and day 16 was significant both in kidney and pylorus ŽMann–Whitney, P - 0.01.. Furthermore, virus titre was significantly lower 44 days after infection compared to 16 days after infection, both in kidney ŽMann–Whitney, P - 0.01. and in pylorus ŽMann–Whitney, P - 0.001.. The frequency of fish with detectable virus Žprevalence. followed the same pattern as the virus titre ŽTable 1., and after 16 days all examined fish had detectable virus in both kidney and pylorus. After 44 days, 50% of the fish still had detectable virus in the kidney, compared to 20% in pylorus. There were no mortality in the infected groups, or in the control groups. In the control groups, weight-specific feed intake ŽFI. increased significantly Ž t-test, P - 0.001. from 5.9 " 0.2 mg gy1 dayy1 Ž n s 60. 9 days after infection, to 10.5 " 0.4 mg gy1 dayy1 Ž n s 50. 44 days after infection. In the infected groups, FI remained between 5 and 8 mg gy1 dayy1 during the course of the experiment ŽFig. 2a.. There were significant differences Ž t-tests. between FI in the control groups and the infected groups, both at day 9 Ž P - 0.01., day 23 Ž P - 0.05., day 30 Ž P - 0.001. and day 44 Ž P - 0.001.. Specific growth rates ŽSGR. in the control groups increased until 30 days after infection, and thereafter SGR stabilised at approximately 1.4% b.w. dayy1 ŽFig. 2b.. After 30 days, SGR in infected fish was generally lower than in uninfected fish, but this difference was only significant at day 30 after infection Ž t-test, P - 0.05.. At the termination of the experiment Ž6 December. the infected fish Ž326.7 " 3.3 g, n s 337. was significantly Ž t-test, P - 0.05. 5% smaller than the uninfected fish Ž343.1 " 5.7 g, n s 90.. Specific growth rate between 20 days before and 44 days after infection Ž3 October to 6 December. was significantly lower Ž t-test, P - 0.001. in
Table 1 Frequency of fish with detectable virus Žprevalence., and virus titres Žp.f.u. gy1 ; mean"S.E.. in kidney and pylorus of Atlantic salmon after infection with IPNV Ž ns10 fish per sample. Virus titre Žp.f.u. gy1 .
Time after infection Ždays.
Prevalence Ž%. Kidney
Pylorus
Kidney
Pylorus
9 14 16 18 23 30 35 39 44
100 100 100 90 90 100 100 89 50
30 80 100 40 100 60 80 78 20
3.6"1.0=10 3 4.6"3.3=10 4 6.3"5.0=10 6 7.6"6.9=10 5 3.6"3.3=10 6 4.0"1.8=10 5 4.3"3.3=10 5 5.4"2.7=10 5 2.7"2.2=10 4
1.1"0.7=10 3 1.5"1.0=10 5 2.4"1.5=10 6 7.7"7.0=10 5 4.0"3.3=10 5 6.1"3.7=10 4 1.5"0.7=10 4 2.3"2.2=10 5 2.2"2.2=10 3
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
189
Fig. 1. Box-and-whisker plot of virus titre Žp.f.u. gy1 . in Ža. kidney and Žb. pylorus of Atlantic salmon after infection with IPNV. The boxes indicate the range of 50% of the observations, while the horizontal lines indicate the medians. Whiskers indicate the range of the observations, with outliers Žbeyond 1.5 times the box height. being denoted by asterisks Ž)..
infected fish Ž1.10 " 0.01% b.wt dayy1 , n s 335. compared to the control fish Ž1.21 " 0.02% b.wt dayy1 , n s 90.. There was, however, no difference in maximum SGR between infected fish ŽFig. 3a. and uninfected fish ŽFig. 3b., but the frequency of fish with low SGR was higher in the groups of infected fish.
Fig. 2. Ža. Weight-specific feed intake ŽFI; mg gy1 dayy1 . and Žb. specific growth rate ŽSGR; % Žb.w. dayy1 .. in IPNV-infected Atlantic salmon Žfilled symbols., compared with uninfected control fish Žopen symbols.. Each data-point represents mean"SE of 46–60 fish.
190
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
Fig. 3. Frequency of specific growth rate ŽSGR; % b.w. dayy1 . in Ža. IPNV-infected Atlantic salmon, and Žb. uninfected control fish, during the period 20 days before and 44 days after infection. The figures represent 335 infected and 90 uninfected fish.
In individual fish with detectable virus, weight-specific feed intake ŽFI. decreased slightly as a function of virus titre Ž V ., according to the regression: FI s y0.228 logV q 8.417 Ž r s 0.23, P s 0.05, n s 82. in kidney ŽFig. 4a., and: FI s y0.316 logV q 9.627 Ž r s 0.29, P - 0.05, n s 59. in pylorus ŽFig. 4b.. The individual variation
Fig. 4. Individual weight-specific feed intake ŽFI; mg gy1 dayy1 . as a function of virus titre Žp.f.u. gy1 . in Ža. kidney and Žb. pylorus. The plots represent all fish with detectable virus from day 9 to day 44 after infection with IPNV.
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
191
in feed intake was, however, large, and some fish had a high infection of IPNV Ž10 6 –10 7 p.f.u. gy1 . without a suppression of the feed intake.
4. Discussion The present results demonstrate that IPNV-infection affects feed intake and growth in Atlantic salmon, but the fish may have a relatively severe infection of virus before any effects on appetite are detectable. In general, feed intake in fish is determined by numerous biotic and abiotic factors, including fish size, age, social behaviour, maturation status, stress, temperature, light, salinity and type of food Žreviewed by, e.g., Brett, 1979; Jobling, 1994.. The estimated feed intake in a group of fish is mainly based upon fish size and water temperature, but several studies have demonstrated a large intrapopulation and day-to-day variation in individual feed intake ŽJobling, 1994.. In addition, a seasonal feeding rhythm, independent of other growth parameters, is reported in salmonids ŽJobling, 1987; Sæther et al., 1996.. Few studies have however monitored feed intake in fish during an outbreak of a disease. Using a self-feeding method, Anthouard and Wolf Ž1988. measured total feed intake in a group of wels, Silurus glanis L., after an outbreak of a branchial ectoparasite ŽDactylogyrus., and found a decline in appetite until the fish recover after treatment. A number of feeding experiments have demonstrate that the X-ray method is well suited for studies of appetite in fish ŽTalbot and Higgins, 1983; Jørgensen and Jobling, 1989; Jobling et al., 1995.. The X-radiographic method enabled us to study the voluntary feed intake in individual fish with known virus titres, without stressing the fish during the feeding period. In general, the effects of illness on appetite may be explained in terms of general impairment, a malfunction of digestion or metabolism, or other mechanisms which inhibit feeding during illness. The physiological control of appetite are, however, complex Žreviewed by Fletcher, 1984., and in the present study, it is difficult to evaluate why some of the infected fish lost appetite. Feed intake is probably under multifactorial control ŽFletcher, 1984., and loss of appetite during illness may have several explanations. In the present study, feed intake for some weeks after infection were slightly lower than normal, both in the infected groups and the control groups, indicating that handling during infection may have suppressed feed intake temporarily. However, this does not explain why the feed intake in all infected groups were significant lower than the control groups, indicating that IPNV significantly affected the appetite in Atlantic salmon. Several studies have documented IPNV in juvenile Že.g., Smail et al., 1992, 1995. and post-smolts of Atlantic salmon Že.g., Christie et al., 1988; Jarp et al., 1995.. Kidney yield IPNV most frequently ŽYamamoto, 1975; Mangunwiryo and Agius, 1988; Wolf, 1988. and in our experiment, virus titre rapidly increased in kidney after infection, and the prevalence and virus titre in kidney remained high during the course of the experiment. Pylorus is, however, the main target organ for IPNV, producing marked pancreatic necrosis ŽMangunwiryo and Agius, 1988; Wolf, 1988.. According to Wolf Ž1988., from 10 6 to 10 9 p.f.u. gy1 may be mortal to juvenile fish. The lethality of IPNV
192
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
is age dependent, and the prevalence of IPNV in carriers of rainbow trout, Oncorhynchus mykiss ŽWalbaum., fluctuated during the year ŽMangunwiryo and Agius, 1988.. In an experimental infection of IPNV in Atlantic salmon, Stangeland et al. Ž1996. found a cumulative mortality between 30 and 60%, while no fish died in our experiment. The fish in their experiment were smaller Žinitial weight 50 g. than in our experiment Žinitial weight 172 g.. In addition, their fish were stressed by lowering the water level in the fish tanks, and the level of plasma Cly indicated that mortality partly may be caused by a poor hypoosmoregulatory capacity. However, the pattern of mortality as a function of the time after infection ŽStangeland et al., 1996. corresponds with the increase in virus titre in the present experiment, indicating that factors such as size, hypoosmoregulatory capacity and stress may have affected mortality and the development of virus titres in our experiment. Despite the fact that IPNV affected feed intake in the present experiment, a low virus-infection does not necessarily have large effects on the growth of the fish. IPNV-infected Atlantic salmon smolts in two farms in Scotland maintained at least 75% of the expected growth during the first post-smolt year, indicating that IPNV does not compromise the digestive function of the pancreas ŽSmail and Munro, 1985.. However, post-smolts with high IPNV titres often have low condition factors ŽSmail et al., 1995., but the causal relationship is still uncertain, because both low condition factor and high virus titres may be consequences of poor hypoosmoregulatory capacity. In summary, our study demonstrated that IPNV did only affect feed intake and growth of highly infected Atlantic salmon. The results indicate that a low infection of IPNV does not pose a serious economic treat to the aquaculture of Atlantic salmon.
Acknowledgements This study was financed by the Norwegian Research Council, project 108925r100 and 122874r122. The authors thanks Sten Siikavuopio, Lill-Heidi Johansen and the staff of the Fish Health Laboratory, Aquaculture Research Station, Tromsø, for technical assistance, and Hilde Toften for comments on the manuscript.
References Anthouard, M., Wolf, V., 1988. A computerized surveillance method based on self-feeding measures in fish population. Aquaculture 71, 151–158. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randal, D.J., Brett, J.R. ŽEds.., Fish Physiology, Vol. 8. Academic Press, New York, pp. 599–675. Christie, K.E., Havarstein, L.S., Djupvik, H.O., Ness, S., Endresen, C., 1988. Characterization of a new ˚ serotype on infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch. Virol. 103, 167–177. Dobos, P., Roberts, T.E., 1983. The molecular biology of infectious pancreatic necrosis virus: a review. Can. J. Microbiol. 29, 377–384. Dulbecco, R., 1980. The nature of viruses. In: Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S. ŽEds.., Microbiology, 3rd edn. Harper and Row Publishers, Philadelphia, pp. 854–884.
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
193
Fletcher, D.J., 1984. The physiological control of appetite in fish. Comp. Biochem. Physiol. 78A, 617–628. Hart, B., 1988. Biological basis of the behaviour of sick animals. Neurosci. Biobehav. Rev. 12, 123–137. Hill, B.J., 1982. Infectious pancreatic necrosis virus and its virulence. In: Roberts, R.J. ŽEd.., Microbial Diseases of Fish. Academic Press, New York, pp. 91–114. Jarp, J., Gjerve, A.G., Olsen, A.B., Bruheim, T., 1995. Risk factors for furunculosis, infectious pancreatic necrosis and mortality in post-smolts of Atlantic salmon Salmo salar L. J. Fish Dis. 18, 67–78. Jobling, M., 1987. Growth of Arctic charr Ž SalÕelinus alpinus L.. under conditions of constant light and temperature. Aquaculture 60, 243–249. Jobling, M., 1994. Fish Bioenergetics. Chapman & Hall, London. Jobling, M., Arnesen, A.M., Baardvik, B.M., Christiansen, J.S., Jørgensen, E.H., 1995. Monitoring feeding behaviour and food intake: methods and applications. Aquaculture Nutr. 1, 131–143. Jørgensen, E., Jobling, M., 1989. Patterns of food intake in Arctic charr SalÕelinus alpinus, monitored by radiography. Aquaculture 81, 155–160. Krogsrud, J., Hastein, T., Rønning, K., 1989. Infectious pancreatic necrosis virus in Norwegian fish farms. In: ˚ Ahne W., Kurstak, E. ŽEds.., Viruses of Lower Vertebrates. Springer, Berlin, pp. 284–291. Mangunwiryo, H., Agius, C., 1988. Studies on the carrier state of infectious pancreatic necrosis virus infections in rainbow trout Salmo gairdeneri Richardson. J. Fish Dis. 11, 125–132. Smail, D.A., Munro, A.L.S., 1985. Infectious pancreatic necrosis virus persistence in farmed Atlantic salmon Ž Salmo salar .. In: Ellis, A.E. ŽEd.., Fish and Shellfish Pathology. Academic Press, New York, pp. 277–288. Smail, D.A., Bruno, D.W., Dear, G., McFarlane, L.A., Ross, K., 1992. Infectious pancreatic necrosis ŽIPN. virus Sp serotype in farmed Atlantic salmon, Salmo salar L., post-smolts associated with mortality and clinical diseases. J. Fish Dis. 15, 77–83. Smail, D.A., McFarlane, L., Bruno, D.W., McVicar, A.H., 1995. The pathology of an IPN-Sp sub-type ŽSh. in farmed Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. J. Fish Dis. 18, 631–638. Snieszko, S.F., Wolf, K., Camper, J.E., Pettijohn, L.L., 1959. Infectious nature of pancreatic necrosis. Trans. Am. Fish. Soc. 88, 289–293. Stangeland, K., Høie, S., Taksdal, T., 1996. Experimental induction of infectious pancreatic necrosis in Atlantic salmon, Salmo salar L., post-smolts. J. Fish Dis. 19, 323–327. Swanson, R.N., Gillespie, J.H., 1979. Pathogenesis of infectious pancreatic necrosis in Atlantic salmon Ž Salmo salar .. J. Fish. Res. Board Can. 36, 587–591. Sæther, B.-S., Johnsen, H.K., Jobling, M., 1996. Seasonal changes in food consumption and growth of Arctic charr exposed to either simulated natural or a 12:12 LD photoperiod at constant water temperature. J. Fish Biol. 48, 1113–1122. Talbot, C., Higgins, P.J., 1983. A radiographic method for feeding studies on fish using metallic iron powder as a marker. J. Fish Biol. 23, 211–220. Wolf, K., 1988. Infectious pancreatic necrosis. In: Wolf, K. ŽEd.., Fish Viruses and Fish Viral Diseases. Comstock Publishing Association, Ithaca, New York, pp. 115–157. Yamamoto, T., 1975. Frequency of detection and survival of infectious pancreatic necrosis virus in a carrier population of brook trout Ž SalÕelinus fontinalis . in a lake. J. Fish. Res. Board Can. 32, 568–570.
Effects of infectious pancreatic necrosis virus ž IPNV/ on appetite and growth in Atlantic salmon, Salmo salar L. B. Damsgard ˚ ) , A. Mortensen, A.-I. Sommer Norwegian Institute of Fisheries and Aquaculture, 9005 Tromsø, Norway Accepted 28 February 1998
Abstract The effects of infectious pancreatic necrosis virus ŽIPNV. infection on appetite and growth in Atlantic salmon, Salmo salar L., were studied under experimental conditions. Eight groups of 60 fish Žage 1 q , initial weight 172 g. were bath infected with IPNV, while two groups served as controls. Virus titres were quantified in kidney and pylorus, both prior to the experiment and eight times from 9 to 44 days after infection. Feed intake of individual fish was measured using X-radiography at sixteen occasions from 7 to 44 days after infection. During the experiment, no mortality occurred in the infected or in the control groups. In infected fish, the virus titre in both kidney and pylorus increased significantly until 16 days after infection, to approximately 10 6 –10 7 p.f.u. gy1 tissue, while no virus was detected in the control groups. Between 16 and 44 days after infection, the virus titre in pylorus decreased significantly to a level of 10 3 –10 4 p.f.u. gy1. From approximately 20 days after infection, feed intake and specific growth rates were significantly lower in infected fish compared to uninfected fish. Few fish completely lost their appetite, and the study revealed that IPNV-infected fish may have relative high virus titres before any changes in appetite or growth could be detected. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Virus; IPNV; Appetite; Feeding; X-ray; Salmo salar
1. Introduction Many animal diseases and infections are accompanied by a loss of appetite ŽHart, 1988., and feed intake and feeding behaviour are thus generally used to indicate the )
Corresponding author. Tel.: q47-77-62-91-60; fax: q47-77-62-91-80; e-mail: [email protected].
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 4 2 - 7
186
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
health status of fish. Feed intake is also one of the most important factors determining the economic outcome of fish farming. Thus, information of changes in appetite is essential to ensure optimal growth and keep feed wastage to a minimum. Infectious pancreatic necrosis virus ŽIPNV. is an aquatic birnavirus ŽDobos and Roberts, 1983.. The virus causes acute infections, often with fatal outcome, primarily in juvenile salmonids ŽSnieszko et al., 1959; Hill, 1982; Wolf, 1988.. The incubation time of IPNV is approximately 2 to 4 weeks, and the mortality may range from zero to 100% in juvenile fish, depending on the serotype encountered, degree of infection, fish age, species, and water temperature Žreviewed by Wolf, 1988.. Several IPN outbreaks in Atlantic salmon, Salmo salar L., with high mortality during smolting and after transfer to sea cages have also been observed ŽKrogsrud et al., 1989; Smail et al., 1992; Smail et al., 1995; Jarp et al., 1995.. The risk that Atlantic salmon develop IPNV in sea water is affected by origin of the smolts, handling stress and geographical locations of the fish farm ŽJarp et al., 1995.. As in other salmonids, Atlantic salmon surviving an outbreak of IPNV may become life-long asymptomatic carriers of the virus ŽSwanson and Gillespie, 1979; Smail and Munro, 1985.. Despite a general observation of appetite loss in IPNV-infected fish, no experimental studies have monitored individual feed intake and growth of infected fish. To evaluate if loss of appetite or growth reductions can be associated with an IPNV infection in Atlantic salmon, we studied the effects of IPNV infection on appetite and growth in post-smolts, using X-radiography to monitor individual feed intake during six weeks after infection.
2. Materials and methods Ten groups of hatchery reared Atlantic salmon Ž1 q , initial weight 172 g, n s 60 in each group. were reared in 500 l tanks in the Fish Health Laboratory at the Aquaculture Research Station in Tromsø, northern Norway Ž708N 198E., from 3 October 1995 onwards. The fish were individually tagged ŽFTF-69, Floy Tag and Manufacturing, Seattle, USA.. Water temperature was maintained at 108C, salinity was held at 10‰, and the photoperiod simulated to match the natural photoperiod in Tromsø. The fish were fed commercial dry feed pellets ŽFelleskjøpet, Sandnes, Norway; FK-Vekst, 4 mm pellets. in excess, using automatic disc feeders. Three weeks after tagging Ž23 October., eight of the groups were infected with IPNV, while two groups were left uninfected, but were otherwise treated identically. The IPNV used for infection was isolated from Atlantic salmon juveniles after a natural outbreak and passage three times in chinook salmon embryo ŽCHSE-214. cells ŽFlow, Herts, England; ATCC no. CRL 1681.. The stock virus preparation contained 2 = 10 8 TCID50 mly1 Žtissue culture infective doses at 50% endpoint., and was stored at y808C. Experimentally IPNV infection was performed by keeping the fish for 4 h at 108C and 10‰ salinity in 100 l oxygen-saturated water, containing about 2 = 10 5 TCID50 mly1 IPNV. At eight times during the experiment Žday 9, 14, 18, 23, 30, 35, 39 and 44 after
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
187
infection., we sampled head kidney and pylorus from 10 fish from each group. The organs were weighed and homogenised into a 10% Žwrv. suspension in minimum essential medium ŽMEM, Gibco, Renfrewshire, Scotland.. The pylorus homogenate was initially centrifuged for 10 min at 2000 = g. The pylorus supernatant and the kidney homogenate were clarified for 15 min at 12 000 rpm in an Eppendorf centrifuge and the supernatants were used for virus titre assay. CHSE-214 were grown at 208C in MEM, supplemented with 1% Žwrv. non-essential amino acids, 1% L-glutamine and antibiotics. For cell growth purposes 8% foetal calf serum ŽFCS, Gibco. was added to the medium, and 2% FCS was used when the cells were virus infected. For virus titre assays, the cells were seeded into 96-well cell culture plates ŽCostar, Cambridge, USA.. Ten-fold dilution of samples were added on to CHSE-214 cells and observed for cytopathic effect in the monolayers after seven days of incubation. TCID50 were calculated according to a method of Reed and Muench, and one TCID50 dose corresponds to 0.70 plaque-forming units Žp.f.u.. ŽDulbecco, 1980.. The minimum detectable titre was 25 p.f.u. gy1 of tissue Žkidney, pylorus.. The prevalence of IPNV was expressed as the frequency of fish with detectable virus. The IPNV carrier condition was investigated by use of kidney homogenates from 20 fish sampled prior to the experimental IPNV infection and in uninfected fish from the control groups throughout the study. The fish had no known history of IPN, and none of these samples showed cytopathic effect after two additional passages on to new CHSE-214 cells. At regular intervals after infection Ždays 7, 9, 11, 14, 16, 18, 21, 23, 28, 30, 32, 35, 37, 39, 42 and 44., we measured feed intake of individual fish using X-radiography ŽTalbot and Higgins, 1983; Jørgensen and Jobling, 1989; Jobling et al., 1995.. The control groups were measured at four occasions during the experiment Ždays 9, 23, 30 and 44.. Each group was randomly measured two times during the experiment. During the X-ray sessions, feed was replaced with pellets of identical nutrient composition and physical qualities, but formulated to contain 1.5% by weight of X-ray dense glass beads Žsize 8.5 Ballotini, Jencons, Leighton Buzzard, Bedfordshire, England.. Fish were anaesthetised using benzocaine, weighed to the nearest 0.1 g, and X-ray photographed ŽTodd Research machine, 3 s exposure, 80 kV, AGFA Structurix D7 film.. Glass beads in the stomach and intestine of the fish were counted, and feed intake in grams were calculated using a calibration curve describing the relationship between numbers of glass beads Ž B . and the weights of feed Ž F; g.: F s 0.013 = B y 0.061 Ž r s 0.99, n s 19, P - 0.001.. Weight-specific feed intake ŽFI. were expressed as mg feed per g fish per day Žmg gy1 dayy1 .. Specific growth rates ŽSGR; % Žb.w. dayy1 .. between two measurements were calculated as: SGR s 100 = Žln W2 y ln W1.rt, where W1 is the weight at time 1, W2 is the weight at time 2, and t is the time in days between measurements. Data are presented as means " standard error ŽS.E... Non-parametric Mann–Whitney U-tests were used to examine for differences in virus titres, while t-tests were used to examine for differences in weight-specific feed intake and specific growth rates. A probability level of less than 0.05 was considered as significant.
188
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
3. Results Prior to the experiment, there were no differences in body weight between the 10 groups, and no virus was detected Žvirus titre - 25 p.f.u. gy1 .. In the control groups, no virus was detected during the experiment Ž9, 23, 30 and 44 days after infection.. In infected fish, the virus titre in both kidney and pylorus increased 2–3 weeks after infection, and thereafter decreased until the end of the experiment ŽTable 1, Fig. 1.. The increase in virus titre between day 9 and day 16 was significant both in kidney and pylorus ŽMann–Whitney, P - 0.01.. Furthermore, virus titre was significantly lower 44 days after infection compared to 16 days after infection, both in kidney ŽMann–Whitney, P - 0.01. and in pylorus ŽMann–Whitney, P - 0.001.. The frequency of fish with detectable virus Žprevalence. followed the same pattern as the virus titre ŽTable 1., and after 16 days all examined fish had detectable virus in both kidney and pylorus. After 44 days, 50% of the fish still had detectable virus in the kidney, compared to 20% in pylorus. There were no mortality in the infected groups, or in the control groups. In the control groups, weight-specific feed intake ŽFI. increased significantly Ž t-test, P - 0.001. from 5.9 " 0.2 mg gy1 dayy1 Ž n s 60. 9 days after infection, to 10.5 " 0.4 mg gy1 dayy1 Ž n s 50. 44 days after infection. In the infected groups, FI remained between 5 and 8 mg gy1 dayy1 during the course of the experiment ŽFig. 2a.. There were significant differences Ž t-tests. between FI in the control groups and the infected groups, both at day 9 Ž P - 0.01., day 23 Ž P - 0.05., day 30 Ž P - 0.001. and day 44 Ž P - 0.001.. Specific growth rates ŽSGR. in the control groups increased until 30 days after infection, and thereafter SGR stabilised at approximately 1.4% b.w. dayy1 ŽFig. 2b.. After 30 days, SGR in infected fish was generally lower than in uninfected fish, but this difference was only significant at day 30 after infection Ž t-test, P - 0.05.. At the termination of the experiment Ž6 December. the infected fish Ž326.7 " 3.3 g, n s 337. was significantly Ž t-test, P - 0.05. 5% smaller than the uninfected fish Ž343.1 " 5.7 g, n s 90.. Specific growth rate between 20 days before and 44 days after infection Ž3 October to 6 December. was significantly lower Ž t-test, P - 0.001. in
Table 1 Frequency of fish with detectable virus Žprevalence., and virus titres Žp.f.u. gy1 ; mean"S.E.. in kidney and pylorus of Atlantic salmon after infection with IPNV Ž ns10 fish per sample. Virus titre Žp.f.u. gy1 .
Time after infection Ždays.
Prevalence Ž%. Kidney
Pylorus
Kidney
Pylorus
9 14 16 18 23 30 35 39 44
100 100 100 90 90 100 100 89 50
30 80 100 40 100 60 80 78 20
3.6"1.0=10 3 4.6"3.3=10 4 6.3"5.0=10 6 7.6"6.9=10 5 3.6"3.3=10 6 4.0"1.8=10 5 4.3"3.3=10 5 5.4"2.7=10 5 2.7"2.2=10 4
1.1"0.7=10 3 1.5"1.0=10 5 2.4"1.5=10 6 7.7"7.0=10 5 4.0"3.3=10 5 6.1"3.7=10 4 1.5"0.7=10 4 2.3"2.2=10 5 2.2"2.2=10 3
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
189
Fig. 1. Box-and-whisker plot of virus titre Žp.f.u. gy1 . in Ža. kidney and Žb. pylorus of Atlantic salmon after infection with IPNV. The boxes indicate the range of 50% of the observations, while the horizontal lines indicate the medians. Whiskers indicate the range of the observations, with outliers Žbeyond 1.5 times the box height. being denoted by asterisks Ž)..
infected fish Ž1.10 " 0.01% b.wt dayy1 , n s 335. compared to the control fish Ž1.21 " 0.02% b.wt dayy1 , n s 90.. There was, however, no difference in maximum SGR between infected fish ŽFig. 3a. and uninfected fish ŽFig. 3b., but the frequency of fish with low SGR was higher in the groups of infected fish.
Fig. 2. Ža. Weight-specific feed intake ŽFI; mg gy1 dayy1 . and Žb. specific growth rate ŽSGR; % Žb.w. dayy1 .. in IPNV-infected Atlantic salmon Žfilled symbols., compared with uninfected control fish Žopen symbols.. Each data-point represents mean"SE of 46–60 fish.
190
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
Fig. 3. Frequency of specific growth rate ŽSGR; % b.w. dayy1 . in Ža. IPNV-infected Atlantic salmon, and Žb. uninfected control fish, during the period 20 days before and 44 days after infection. The figures represent 335 infected and 90 uninfected fish.
In individual fish with detectable virus, weight-specific feed intake ŽFI. decreased slightly as a function of virus titre Ž V ., according to the regression: FI s y0.228 logV q 8.417 Ž r s 0.23, P s 0.05, n s 82. in kidney ŽFig. 4a., and: FI s y0.316 logV q 9.627 Ž r s 0.29, P - 0.05, n s 59. in pylorus ŽFig. 4b.. The individual variation
Fig. 4. Individual weight-specific feed intake ŽFI; mg gy1 dayy1 . as a function of virus titre Žp.f.u. gy1 . in Ža. kidney and Žb. pylorus. The plots represent all fish with detectable virus from day 9 to day 44 after infection with IPNV.
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
191
in feed intake was, however, large, and some fish had a high infection of IPNV Ž10 6 –10 7 p.f.u. gy1 . without a suppression of the feed intake.
4. Discussion The present results demonstrate that IPNV-infection affects feed intake and growth in Atlantic salmon, but the fish may have a relatively severe infection of virus before any effects on appetite are detectable. In general, feed intake in fish is determined by numerous biotic and abiotic factors, including fish size, age, social behaviour, maturation status, stress, temperature, light, salinity and type of food Žreviewed by, e.g., Brett, 1979; Jobling, 1994.. The estimated feed intake in a group of fish is mainly based upon fish size and water temperature, but several studies have demonstrated a large intrapopulation and day-to-day variation in individual feed intake ŽJobling, 1994.. In addition, a seasonal feeding rhythm, independent of other growth parameters, is reported in salmonids ŽJobling, 1987; Sæther et al., 1996.. Few studies have however monitored feed intake in fish during an outbreak of a disease. Using a self-feeding method, Anthouard and Wolf Ž1988. measured total feed intake in a group of wels, Silurus glanis L., after an outbreak of a branchial ectoparasite ŽDactylogyrus., and found a decline in appetite until the fish recover after treatment. A number of feeding experiments have demonstrate that the X-ray method is well suited for studies of appetite in fish ŽTalbot and Higgins, 1983; Jørgensen and Jobling, 1989; Jobling et al., 1995.. The X-radiographic method enabled us to study the voluntary feed intake in individual fish with known virus titres, without stressing the fish during the feeding period. In general, the effects of illness on appetite may be explained in terms of general impairment, a malfunction of digestion or metabolism, or other mechanisms which inhibit feeding during illness. The physiological control of appetite are, however, complex Žreviewed by Fletcher, 1984., and in the present study, it is difficult to evaluate why some of the infected fish lost appetite. Feed intake is probably under multifactorial control ŽFletcher, 1984., and loss of appetite during illness may have several explanations. In the present study, feed intake for some weeks after infection were slightly lower than normal, both in the infected groups and the control groups, indicating that handling during infection may have suppressed feed intake temporarily. However, this does not explain why the feed intake in all infected groups were significant lower than the control groups, indicating that IPNV significantly affected the appetite in Atlantic salmon. Several studies have documented IPNV in juvenile Že.g., Smail et al., 1992, 1995. and post-smolts of Atlantic salmon Že.g., Christie et al., 1988; Jarp et al., 1995.. Kidney yield IPNV most frequently ŽYamamoto, 1975; Mangunwiryo and Agius, 1988; Wolf, 1988. and in our experiment, virus titre rapidly increased in kidney after infection, and the prevalence and virus titre in kidney remained high during the course of the experiment. Pylorus is, however, the main target organ for IPNV, producing marked pancreatic necrosis ŽMangunwiryo and Agius, 1988; Wolf, 1988.. According to Wolf Ž1988., from 10 6 to 10 9 p.f.u. gy1 may be mortal to juvenile fish. The lethality of IPNV
192
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
is age dependent, and the prevalence of IPNV in carriers of rainbow trout, Oncorhynchus mykiss ŽWalbaum., fluctuated during the year ŽMangunwiryo and Agius, 1988.. In an experimental infection of IPNV in Atlantic salmon, Stangeland et al. Ž1996. found a cumulative mortality between 30 and 60%, while no fish died in our experiment. The fish in their experiment were smaller Žinitial weight 50 g. than in our experiment Žinitial weight 172 g.. In addition, their fish were stressed by lowering the water level in the fish tanks, and the level of plasma Cly indicated that mortality partly may be caused by a poor hypoosmoregulatory capacity. However, the pattern of mortality as a function of the time after infection ŽStangeland et al., 1996. corresponds with the increase in virus titre in the present experiment, indicating that factors such as size, hypoosmoregulatory capacity and stress may have affected mortality and the development of virus titres in our experiment. Despite the fact that IPNV affected feed intake in the present experiment, a low virus-infection does not necessarily have large effects on the growth of the fish. IPNV-infected Atlantic salmon smolts in two farms in Scotland maintained at least 75% of the expected growth during the first post-smolt year, indicating that IPNV does not compromise the digestive function of the pancreas ŽSmail and Munro, 1985.. However, post-smolts with high IPNV titres often have low condition factors ŽSmail et al., 1995., but the causal relationship is still uncertain, because both low condition factor and high virus titres may be consequences of poor hypoosmoregulatory capacity. In summary, our study demonstrated that IPNV did only affect feed intake and growth of highly infected Atlantic salmon. The results indicate that a low infection of IPNV does not pose a serious economic treat to the aquaculture of Atlantic salmon.
Acknowledgements This study was financed by the Norwegian Research Council, project 108925r100 and 122874r122. The authors thanks Sten Siikavuopio, Lill-Heidi Johansen and the staff of the Fish Health Laboratory, Aquaculture Research Station, Tromsø, for technical assistance, and Hilde Toften for comments on the manuscript.
References Anthouard, M., Wolf, V., 1988. A computerized surveillance method based on self-feeding measures in fish population. Aquaculture 71, 151–158. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randal, D.J., Brett, J.R. ŽEds.., Fish Physiology, Vol. 8. Academic Press, New York, pp. 599–675. Christie, K.E., Havarstein, L.S., Djupvik, H.O., Ness, S., Endresen, C., 1988. Characterization of a new ˚ serotype on infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch. Virol. 103, 167–177. Dobos, P., Roberts, T.E., 1983. The molecular biology of infectious pancreatic necrosis virus: a review. Can. J. Microbiol. 29, 377–384. Dulbecco, R., 1980. The nature of viruses. In: Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S. ŽEds.., Microbiology, 3rd edn. Harper and Row Publishers, Philadelphia, pp. 854–884.
B. Damsgard ˚ et al.r Aquaculture 163 (1998) 185–193
193
Fletcher, D.J., 1984. The physiological control of appetite in fish. Comp. Biochem. Physiol. 78A, 617–628. Hart, B., 1988. Biological basis of the behaviour of sick animals. Neurosci. Biobehav. Rev. 12, 123–137. Hill, B.J., 1982. Infectious pancreatic necrosis virus and its virulence. In: Roberts, R.J. ŽEd.., Microbial Diseases of Fish. Academic Press, New York, pp. 91–114. Jarp, J., Gjerve, A.G., Olsen, A.B., Bruheim, T., 1995. Risk factors for furunculosis, infectious pancreatic necrosis and mortality in post-smolts of Atlantic salmon Salmo salar L. J. Fish Dis. 18, 67–78. Jobling, M., 1987. Growth of Arctic charr Ž SalÕelinus alpinus L.. under conditions of constant light and temperature. Aquaculture 60, 243–249. Jobling, M., 1994. Fish Bioenergetics. Chapman & Hall, London. Jobling, M., Arnesen, A.M., Baardvik, B.M., Christiansen, J.S., Jørgensen, E.H., 1995. Monitoring feeding behaviour and food intake: methods and applications. Aquaculture Nutr. 1, 131–143. Jørgensen, E., Jobling, M., 1989. Patterns of food intake in Arctic charr SalÕelinus alpinus, monitored by radiography. Aquaculture 81, 155–160. Krogsrud, J., Hastein, T., Rønning, K., 1989. Infectious pancreatic necrosis virus in Norwegian fish farms. In: ˚ Ahne W., Kurstak, E. ŽEds.., Viruses of Lower Vertebrates. Springer, Berlin, pp. 284–291. Mangunwiryo, H., Agius, C., 1988. Studies on the carrier state of infectious pancreatic necrosis virus infections in rainbow trout Salmo gairdeneri Richardson. J. Fish Dis. 11, 125–132. Smail, D.A., Munro, A.L.S., 1985. Infectious pancreatic necrosis virus persistence in farmed Atlantic salmon Ž Salmo salar .. In: Ellis, A.E. ŽEd.., Fish and Shellfish Pathology. Academic Press, New York, pp. 277–288. Smail, D.A., Bruno, D.W., Dear, G., McFarlane, L.A., Ross, K., 1992. Infectious pancreatic necrosis ŽIPN. virus Sp serotype in farmed Atlantic salmon, Salmo salar L., post-smolts associated with mortality and clinical diseases. J. Fish Dis. 15, 77–83. Smail, D.A., McFarlane, L., Bruno, D.W., McVicar, A.H., 1995. The pathology of an IPN-Sp sub-type ŽSh. in farmed Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. J. Fish Dis. 18, 631–638. Snieszko, S.F., Wolf, K., Camper, J.E., Pettijohn, L.L., 1959. Infectious nature of pancreatic necrosis. Trans. Am. Fish. Soc. 88, 289–293. Stangeland, K., Høie, S., Taksdal, T., 1996. Experimental induction of infectious pancreatic necrosis in Atlantic salmon, Salmo salar L., post-smolts. J. Fish Dis. 19, 323–327. Swanson, R.N., Gillespie, J.H., 1979. Pathogenesis of infectious pancreatic necrosis in Atlantic salmon Ž Salmo salar .. J. Fish. Res. Board Can. 36, 587–591. Sæther, B.-S., Johnsen, H.K., Jobling, M., 1996. Seasonal changes in food consumption and growth of Arctic charr exposed to either simulated natural or a 12:12 LD photoperiod at constant water temperature. J. Fish Biol. 48, 1113–1122. Talbot, C., Higgins, P.J., 1983. A radiographic method for feeding studies on fish using metallic iron powder as a marker. J. Fish Biol. 23, 211–220. Wolf, K., 1988. Infectious pancreatic necrosis. In: Wolf, K. ŽEd.., Fish Viruses and Fish Viral Diseases. Comstock Publishing Association, Ithaca, New York, pp. 115–157. Yamamoto, T., 1975. Frequency of detection and survival of infectious pancreatic necrosis virus in a carrier population of brook trout Ž SalÕelinus fontinalis . in a lake. J. Fish. Res. Board Can. 32, 568–570.