Evaluation of a nondestructive diagnostic test for Kudoa thyrsites in farmed Atlantic salmon (Salmo salar)

Aquaculture ELSEVIER

Aquaculture

156 (1997) 139-144

Evaluation of a nondestructive diagnostic test for Kudoa thyrsites in farmed Atlantic salmon ( Sa2m0

sazar)

Sophie St-Hilaire a,b,*, Carl Ribble a, David J. Whitaker Michael L. Kent b

b,

Western College of Veterinuq Medicine, lJnirer.sity ofSaskatchewan, 52 Campus drive, Saskatoon. Saskatchewan S7N 5B4, Canada of Fisheries and Oceans, Pacific Biological Station. Nanaimo, British Columbia V9R 5K6, Canada

’ Department of Herd Medicine,

’ Department

Accepted

8 September

1996

Abstract with Kudoa thyrsites infections is a in the Pacific Northwest. Infection with this parasite is not macroscopically visible in salmon, and thus infected fish are not readily detectable on the processing line. Identification of infected fish relies on either histological or whole-mount evaluation of muscle tissue. A nondestructive, inexpensive diagnostic test for detection of K. thyrsites infection in the musculature of Atlantic salmon was evaluated in this study. The results indicated that the presence or absence of K. thyrsites in the hyohyoideus ventralis muscle of the operculum, as detected in wet-mount preparations, is a good indicator of the presence or absence of the parasite in the body musculature (fillets) of Atlantic salmon. The sensitivity and specificity of the diagnostic test were 79.0% and 94.6%, respectively. The sensitivity of the test was increased to 93% when the test was used to detect only heavily infected fillets. Therefore the presence or absence of K. thyrsites spores in the hyohyoideus centralis muscle is a good indicator of heavily infected fish fillets, but is slightly less accurate at detecting fillets with lighter infections of K. thymites. 0 1997 Elsevier Science B.V. Postharvest

concern

Keywords:

myoliquefaction

to the Atlantic

salmon

(soft

(S&no

Kudos thymites; Diagnostic

* Corresponding

flesh)

s&r)

associated

industry

test; Atlantic salmon

author. Tel.: + 1 306 9667168;

fax: +

I 306 9667159; e-mail: ribbleOadmin3.usask.ca

0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)0008 I- I

140

S. St-Hilaire et al./Aquaculture

156 (19971 139-144

1. Introduction Kudos thyrsites is a saltwater myxosporean that infects the somatic musculature of several species of marine fish on the west coast of North America including: Pacific hake (Merluccius productus), pollock (Theragra chalcogramma), turbot ( Atherestes stomias), petrale sole (Eopsetta jordani), rock sole (Pleuronectes bilineatus), lemon sole (Pleuronectes vet&us), dover sole (Microstomus pacificus), halibut (Hippoglossus stenolepis), starry flounder (Pleuronectes stellarus), coho salmon (Oncorhynchus kisutch), steelhead salmon (Oncorhynchus mykiss), chinook salmon (Oncorhynchus tshawytscha), pink salmon (Oncorhynchus gorbuscha) (Whitaker et al., 1994), tube snout ( Aulorhynchus Juvidus) (Shaw et al., 1997) and lingcod (Ophiodon elongatus) (J.D.W. Moran, Simon Fraser University, pers. comm.). In several of these susceptible species of fish, K. thyrsites has been associated with increased postmortem muscle autolysis (Whitaker et al., 19941, which greatly reduces the market value of the fish. Recently, cultured Atlantic salmon (Sulmo s&r) in the Pacific Northwest with severe postmortem autolysis or ‘soft flesh’ were found to be infected with K. thyrsites (Harrell and Scott, 1985; Whitaker and Kent, 1991). K. thyrsites-associated ‘soft flesh’ in Atlantic salmon has also been reported in Europe (Barja and Toranzo, 1993). These reports have raised concerns in the Atlantic salmon aquaculture industry and prompted interest in the development of methods for identifying infected fish prior to their sale in the market place. Unfortunately, K. thyrsites is not grossly visible in Atlantic salmon. Infections are usually identified by microscopy, either in wet mount preparations or histological sections of muscle tissues. J. McKay (Canadian Aquastart, Victoria, B.C., pers. comm.) and C. Rymes (independent veterinarian, Vancouver, B.C., pers. comm.) have examined the hyohyoideus ventrulis (Harder, 1975) muscle from under the operculum (Fig. 1) for the presence or absence of K. thyrsites spores as an indicator of infection in the body musculature of fish. This technique has the advantage over examining the body musculature directly in that the fish is not disfigured and therefore its market value is not decreased. However, interpretation of results from this test is difficult because no data on the sensitivity and specificity of this sampling technique has been reported. The purpose of this study was to determine the accuracy of this inexpensive, nondestructive diagnostic test for K. thyrsites infection in the body musculature of Atlantic salmon.

m. hyohyoid.vemmlis

Fig. 1. Illustration of the hyoh~oideus w?trah muscle in fish (illustrated by J. Deubner, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada). Point of origin and insertion of this muscle are the ventral portion of the hyoid arch and the lower radii brdnchiostegi of the operculum.

S. St-Hilaire et al./Aquaculture

156 (1997) 139-144

141

2. Materials and methods Two populations of fish were used to evaluate the opercular test. The first group of fish consisted of 104 postsmolt Atlantic salmon (40-120 g> provided by the Pacific Biological Station, Department of Fisheries and Oceans, Nanaimo, B.C. The second group of fish consisted of 42 market-sized Atlantic salmon (2.5 to 5.5 kg), donated by the B.C. Atlantic salmon industry.

3. Experimental

protocol

Prior to sampling, fish were rinsed under tap water. The hyohyoideus uentralis muscle was dissected from under the operculum (Fig. 1). This muscle was taken from both the right and the left opercula in the fish weighing less than 1 kg and from the left operculum in the larger fish. The weight of the muscle samples collected ranged from 0.1 to 0.4 g, depending on the size of the fish sampled. 0.6 to 0.8 ml of saline were added to the muscle, which was then minced with a scalpel blade and pressed between two pieces of plexiglass. The fluid was collected in a test tube and let to stand for 30 min at room temperature. Two wet-mount slides were made using a 22 mm’ coverslip and approximately 30 pl of material pipetted from the bottom of the test tubes where the minced tissue had settled. The slides were examined under 125 X phase contrast microscopy for K. thyrsites spores using an ‘S’-shaped search pattern. A sample was considered positive if K. thyrsites spores were observed on either of the two slides examined. A sample of body muscle weighing a total of 4.0 g was collected from the lateral craniodorsal, median lateral and caudal regions of the right side of those fish weighing less than 1 kg. To facilitate mincing, the sample was divided into two parts each weighing approximately 2 g 3 ml of saline were added to each 2 g of tissue. The sample was then thoroughly minced with a scalpel blade and pressed between two plexiglass plates. The fluid from both 2 g samples was combined into one test tube. Two wet-mount slides were made from each test tube sample and examined as above. To determine whether the body musculature of the market-sized fish was infected with K. thymites, a total of 40 g of muscles was dissected from the right side of the fish. The same regions were sampled as with the smaller fish. The samples from the three regions of the fish musculature sampled were combined and a 24 g subsample was mixed with 10 ml of saline, and homogenized with a Stomacher Lab Blender 80 (Seaward, London, UK) for 3 to 5 min. A 5 g subsample was taken from the blender and combined with 100 ml of 0.5% trypsin solution for 1 h. The sample was then centrifuged for 15 min at 4000 ‘pm, and the supematant was decanted. The pellet was resuspended in 3 ml of saline. The sample was examined as above.

4. Analysis of data A total of 146 fish were sampled in this study. Based on a subjective evaluation of the number of spores present on each slide, the intensity of K. thyrsites infection of the

142

S. St-Hilaire et al./Aquaculture

Table 1 Intensity of K. thy-sitesinfection in the hyohyoideus intensity of infection in the fillet of fish Opercular

muscle

Heavily infected Lightly infected Not infected Total

156 (1997) 139-144

wntrulis

muscle

(opercular muscle) compared to the

Fillet Heavily infected

Lightly infected

Not infected

Total

17 9 2 28

1 16 9 26

0 6 86 92

18 30 98 146

body musculature and the hyohyoideus ventralis muscle of each fish was categorized into not infected, lightly infected or heavily infected. Fish were considered lightly infected if less than 10 spores were observed per slide. The agreement between the two different levels of infection was determined by calculating for K (Norman and Streiner, 1994). The two intensities of infection for the body muscle and the opercular muscle were grouped together to calculate the sensitivity and specificity of using the hyohyoideus c.entralis as a predictor of K. thyrsites in the body musculature of fish (Martin et al., 1987). These two measures were calculated using the computer program EpiInfo 6.0 (Center for Disease Control, Atlanta, GA).

5. Results The intensity of K. thyrsites infection in the hyohyoideus ventralis muscle is compared to the intensity of infection in the fillet muscle of fish in Table 1. The sensitivity and specificity of the diagnostic test for all fish evaluated and the 95% confidence interval for these measurements were 79% (66%, 89%) and 93% (87%, 98%) respectively. The sensitivity and specificity, when only the fish weighing less than 1 kg were analyzed, were 80% (63%, 91%) and 93% (87%, 99%) respectively. The sensitivity and specificity for the market fish were 79% (54%, 93%) and 87% (65%, 97%) respectively. The sensitivity and specificity of the test when it was used on heavily infected fish were 93% (75%, 99%) and 93% (86%, 97%) respectively. Significant agreement was found between the intensities of K. thyrsites infections in the body musculature and the hyohyoideus ventralis muscles of fish (K = 0.55, p < 0.001).

6. Discussion The presence or absence of K. thyrsites spores in the hyohyoideus ventralis muscle was an accurate indicator of the presence or absence of the parasite in the body musculature of Atlantic salmon. Using this opercular muscle to screen fish for K. thyrsites provided a nondestructive method of assessing fish for the presence of a parasite that was otherwise difficult to detect without sampling a large amount of fillet

S. St-Hilaire et al./Ayuaculture

156 (1997) 139-144

143

muscle. The sensitivity and specificity of the opercular muscle test were found to be consistently good despite the variation in size of the test population and minor changes in the test protocol, suggesting that it was a robust and accurate method of detecting K. thyrsites in Atlantic salmon fillets. This study had few false positive fish (infected hyohyoideus ventralis muscle but uninfected body musculature) indicating this diagnostic test has a high specificity. The identification of K. thyrsites spores in the body muscle of fish was pathognomonic for the parasite in this tissue; however, with the technique we used, it is possible that some light fillet infections were not detected. In addition, prespore stages of the parasite cannot be detected with wet-mount preparations. Therefore, missed light infections of K. thyrsites in the fillet or presporogenic stages of the parasite may account for the false positive fish identified, and thus we believe our estimate of the specificity of the opercular muscle test is a conservative one. The sensitivity of the opercular test in detecting fillet infections was slightly lower than its specificity, but it was consistent regardless of the size of the fish examined. Nine of the 11 false negative fish (infected body musculature but uninfected hyohyoideus ventralis muscle) had lightly infected fillets (Table 1). We found that fish with light infections of K. thyrsites do not show noticeable signs of muscle deterioration after harvest (St-Hilaire, 1996). In heavily infected fillets, the sensitivity of the opercular test was 93%. The test should therefore be useful in identifying K. thyrsites infections that are intense enough to potentially cause ‘soft flesh.’ An important limiting factor of the opercular muscle test was the time required to perform the test. On average each opercular muscle required 20 min to evaluate. This makes the test impractical for large scale screenings of individual fish for K. thyrsites in the processing line. However, the opercular muscle test would be useful for estimating the prevalence of K. thyrsites infections in populations of fish either at the farm or at the processing plant. In this case, only a proportion of the fish would be sampled and the time required to evaluate these fish would therefore be reasonable.

7. Conclusion The opercular muscle test evaluated in this study can be used to identify groups of fish at high risk for developing K. thyrsites- associated ‘soft flesh,’ and thus could assist the aquaculture industry with marketing decisions.

Acknowledgements This work was funded by the B.C. Salmon Farmers Association, the Department of Fisheries and Oceans, and the Department of Herd Medicine (WCVM) at the University of Saskatchewan. The diagnostic test evaluated in this study was developed by Joan McKay (Canadian Aquastart, Victoria, B.C.) and Carolyn Rymes (Vancouver, B.C.) independently.

144

S. St-Hilaire et al./Aquaculture

156 (1997) 139-144

References Barja, J.L., Toranzo, A.E., 1993. Myoliquefaction postmortem caused by the myxosporean Kudon thyrsites in reared Atlantic salmon in Spain. Bull. Eur. Assoc. Fish Pathol. 13 (31, 86-88. Harder, W., 1975. Anatomy of Fishes. E. Schweiaerbartsche Verlagsbuchhandlung, Stuttgart, p. 109. Harrell, L.W., Scott, T.M., 1985. Kudos thyrsites (Gilchrist) (myxosporea: multivavulida) in Atlantic salmon, Salmo sular. J. Fish Dis. 8, 329-332. Martin, S.W., Meek, A.H., Willeberg, P., 1987. Veterinary Epidemiology Principles and Methods, vol. 63. Iowa State University Press, Ames, IA, pp. 275-277. Norman, G.R., Streiner, D.L., 1994. Biostatistics. The Bare Essentials. Mosby, St. Louis, MI, pp. 164-167. Shaw, R.W., Hervio, D.M.L., Devlin, R.H., Adamson, M.L., 1997. Infection of Aulorhynchusflaridus (Gill) (osteichthyes: gasterosteiformes) by Kudos thyrsites (Gilchrist) (myxosporea: multivalvulida). J. Parasitol. (in press). St-Hilaire, S., 1996. The Epidemiology of Kudos thyrsites in Atlantic salmon (Salvo s&r). MS. thesis, University of Saskatchewan, Saskatoon, Saskatchewan, pp. 3 l-46. Whitaker, D.J., Kent, M.L., 1991. Myxosporean Kudos thyrsites: A cause of soft flesh in farm-reared Atlantic salmon. J. Aquat. An. Health. 3, 291-294. Whitaker, D.J., Kent, M.L., Margolis, L., 1994. Myxosporean parasites and their potential impact on the aquaculture industry, with emphasis on Kudon species. Kudos workshop proceedings, Province of British Columbia, Ministry of Agriculture, Fisheries and Food, pp. 2-7.