Soft-flesh suppression technology: Inhibiting the post-harvest effects of Kudoa thyrsites infection in farmed Atlantic salmon

Table of contents

• Executive Summary
• Introduction
• Industry challenge being addressed
• Innovation technology being tested for the industry
• Objective
• Deliverables
• Analysis of laboratory scale HHP tested strategies for pilot industrial scale application
• Pilot HHP technology application at selected fish farm site
• Evaluation of industrial pilot project results
• Cost/ benefit analysis of industrial scale soft-flesh suppression technology application
• Recommendations for commercialization and implementation
• Future Directions
• Acknowledgements

Executive Summary

Soft-flesh syndrome presents a significant challenge to the fish-farming industry by compromising product quality and lending to a negative consumer stigma of farmed fish products. In farm-reared Atlantic salmon (Salmo salar) the most common cause of soft-flesh is a parasitic infection caused by Kudoa thyrsites. There are no available treatments for K. thyrsites infection and a technology is urgently needed to suppress the damage invoked by this parasite on fish flesh after harvesting. Implementation of a modified existing food processing technology to suppress K. thyrsites in fish flesh would immediately increase productivity and sustainability of the salmon farming industry. To suppress the manifestation of soft-flesh, a pilot industrial trial of high hydrostatic pressure processing (HHP) was implemented as this technology has been successful at controlling parasite infestation in other meat processing industries. This project successfully tested the viability of HHP technology for suppressing the manifestation of soft-flesh. However, the results of this pilot study demonstrate 1) the colour of salmon flesh is highly sensitive to pressure treatment and product quality is adversely affected, and 2) HHP was not effective at suppressing the post-harvest effects of K. thyrsites infection in farmed Atlantic salmon.

Introduction

Industry challenge being addressed

Soft-flesh syndrome presents a significant challenge to the fish-farming industry by compromising product quality and lending to a negative retailer and consumer stigma of farmed fish products. One of the causative agents of soft-flesh syndrome is a fish parasite (Kudoa thyrsites). K. thyrsites affects a broad range of fish species worldwide, yet little is known about the infectivity of the parasite thereby making it very difficult to control. Consequently, K. thyrsites infection can result in substantial economic losses to finfish harvest industries worldwide. For Campbell River production sites alone, Marine Harvest Canada’s estimates a 10% loss of annual yield attributed to soft-flesh syndrome after harvest.

The complete lifecycle of K. thyrsites is not known. It is a Myxosporean parasite, possibly has more than one host stage, and is not a risk to humans. Furthermore, it is not known how the parasite migrates to the site of infection in the muscle fibers of fish. However, once in a muscle fiber of the host a pseudocyst forms around the parasite in which spore stages develop. Unless fish are severely infected there is no obvious pathology of K. thyrsites infection in live or freshly harvested fish. However, when the host dies, over the next several days the pseudocysts containing myxospores break down, releasing proteolytic enzymes that digest the surrounding muscle fibers and produce “soft-flesh” (myoliquefaction). At low levels of infection the pathology of K. thyrsites infection can go unnoticed in commercially sold fish, but at higher levels of infection the proteolytic activity produces extensive myoliquefaction and the fish meat is no longer viable commercially. The effects of the parasite are most devastating to fish marketed as fresh or cold-smoked. However, because of its colour and texture, the post mortem effects of K. thyrsites infection are especially pronouncedon salmon flesh, particularly that of farmed Atlantic salmon. Because so little is known about the biology of this parasite there are no available treatments. Until K. thyrsites infections can be prevented, a technology is urgently needed to maintain the quality of harvested fish and reduce the economic impact of K. thyrsites on the B.C. salmon farming industry. This innovation proposal was designed to evaluate a potential industry-ready parasite suppression technology to minimize the deleterious effects of K. thyrsites infection on farmed Atlantic salmon after harvesting.

Innovation technology being tested for the industry

The effect of K. thyrsites infection on harvested salmon is an urgent problem in Canada’s aquaculture industry. Currently there are no technological innovations to limit the effects of K. thyrsites expression in fish post-harvest. B.C. salmon farmers in particular require an immediate solution that will limit the damaging effects of parasite infection and conserve the highest possible quality of fresh product to be available to retailers and consumers. Until a means of preventing K. thyrsites infection is developed, a processing technology is needed for immediate application to farmed salmon processing. After harvesting, fish maintained at a slightly lower pre-freeze chilled storage temperature (0-2ºC) show a lower degree of parasite pathology than fish arriving at warmer temperatures (6-8 ºC). However, the slight decrease in the rate of parasite expression by cooler transport temperatures without freezing is inadequate; therefore a more potent, but food safe technology needs to be identified. One of the strongest candidate food processing technologies for application towards K. thyrsites suppression in harvested salmon is high hydrostatic pressure processing to neutralize the proteolytic activity that causes myoliquefaction.

High hydrostatic pressure processing (HHP) is widely accepted as an effective means of preserving foods without the use of chemicals or heat-denaturation of the product. HHP processing is already used for a range of applications in the seafood industry including increased shellfish processing efficiency and improving the microbiological shelf-life of shellfish and finfish products. HHP has also been tested as an effective treatment for parasite inactivation in meat products, including fish. However, the current protocol for parasite inactivation affects the quality of fresh fish product. Because K. thyrsites is not a human pathogen, a milder HHP treatment may be appropriate. However, the effectiveness of HHP processing needs to be tested to develop a protocol sufficient to suppress K. thyrsites expression in infected fish while maintaining the highest possible quality of fresh fish.

Objective

The objective of this project was to evaluate a pilot industrial application of high hydrostatic pressure technology for limiting soft-flesh manifestation (pit formation) to less than 20 pits per 100 gram (≤K2 grade) with acceptable colour to preserve market value according to parameters defined from laboratory analysis.

Deliverables

Analysis of laboratory scale HHP tested strategies for pilot industrial scale application

The objective of this deliverable was to assess which treatment regimes did not drastically affect the market quality of the fillet. Fish fillet quality parameters such as colour, texture, flesh integrity (gaping), and smell were evaluated in pressure treated and untreated 150-250 g fillet portions using standard operation procedures for quality control at Marine Harvest Canada. In addition, a digital colour analyzer (Minolta Chromameter) was used for measuring changes in fish fillet colour. HHP treatments significantly affecting fillet quality would be unacceptable for commercial purposes, and therefore would be invalid even if they could control myoliquefaction. Based on this preliminary stage, treatments less than 40k psi were deemed as acceptable for pilot industrial application using whole fish.

Pilot HHP technology application at selected fish farm site

For this component of the trial the fish were collected from the Okisollo farm site as fish from this site historically have a high percentage of Kudoa manifestation (10-15%). The fish were manually harvested (seined and stunned) as the testing occurred outside the regular site harvest schedule. Samples were transported by boat to Campbell River and then by truck to Port Hardy for treatment and processing. Pressure treatments ranging from 20k psi-35 k psi were applied for durations of 30 to 180 seconds on whole fish after harvesting. Whole fish were loaded into the HHP machine for treatment, unloaded, then treated and untreated (control) fish were manually filleted, and qualitative analyses were conducted. Fillets were individually packaged and labelled for tracking product quality. At the end of fish processing and fillet analysis on day 1 the samples were transported to the BC Centre for Aquatic Health Sciences (BC CAHS) in Campbell River for monitoring and further evaluation of pressure treatment effects and parasite manifestation on day 3 and day 5.

Evaluation of industrial pilot project results

The pilot tests using fillets (pressure and processing time investigated: 20, 25 and 30 thousand lb/in2 (k psi) for 30, 60, 90 and 120 seconds) revealed that lower and higher levels of HHP affected colour independent of the time of exposure. The change in color was measured using a colorimeter (Chromameter) and also through visual inspection with industry-standard colour index cards. The numerical data of change in color obtained with the chromameter was consistent with the visual information. The fillets became pale, indicating loss of pigmentation, and were below the market standard for the product, and therefore would not have any commercial value. In addition, it would be both logistically and very cost ineffective to apply the treatment in fillets. Consequently, no further tests were carried out using fillets and the pilot test advanced to whole fish assays. The initial find-range studies applying the same treatments stated above but using whole fish, indicated the HHP values and time of exposure that should be pursued to evaluate the utilization of this technology to control myoliquefaction due to Kudoa thyrsites infection were: 20000 lb/in2 for 30 seconds (the lowest possible treatment for the HHP machine), 20000 lb/in2 60, 90 and 120 seconds, and 25000 and 30000 lb/in2 for 30 and 60 seconds. Colour change was still detected in the fillets even at this low pressure treatment range.

To evaluate the effectiveness of HHP treatment for suppressing myoliquefaction for trials 3-5 individual fillets were surveyed for the presence and abundance of proteolytic lesions (pits) in the muscle tissue. These trials were conducted using pre-harvest size fish from a production site with a historically higher incidence of K. thyrsites infestation. As the fish were needed, a simulated harvest was conducted using a box seine and manually percussive stunning each fish and transporting them on ice to the processing plant to conduct the pressure treatment trials. These fish were slightly paler than the fillets used in the first trial as the fish were not as old or pigmented. However, the same colour change pattern was observed.

To determine the effects of HHP treatments on the fish flesh, pH was also monitored as lower pH is associated with increased proteolytic activity associated with K. thyrsites manifestation. The pH of all pressure treated fillets (except 30 K psi for 60 seconds) remained the same or slightly higher (closer to neutral) than did the pH of the controls.  To assess the effects of higher muscle pH (less stressed) at harvest, fish on trial 5 were anaesthetized (tricaine methanesulfonate – TMS) prior to harvesting and subjected to the following treatments: 20000 lb/in2 for, 90, 120, and 180 seconds: 25000 lb/in2 for 30 seconds. The pH of all the anaesthetized fish was higher than in the non- anaesthetized fish.

For trials 2 through 5, we also monitored the manifestation of K. thyrsites by quantifying the number of pits and extent of myoliquefaction in the fillets in response to HHP treatments. However, even the highest treatment with marginal product quality parameters (30,000 lb/in2 for 60 seconds) did not control myoliquefaction. For trials 4 and 5, we then further narrowed down the treatments tested to be able to increase the number of fish per treatment (10 fish per treatment were used in this test). The following treatments were tested: 20000 lb/in2 for 30, 60, 90, 120, and 180 seconds: 25000 lb/in2 for 30 seconds. Again the results demonstrated that myoliquefaction occurred in all treatments, independent of HHP pressure intensity or duration of exposure. These results demonstrated that HHP adversely affected the color and did not suppress myoliquefaction caused by Kudoa thyrsites infection.

Cost/ benefit analysis of industrial scale soft-flesh suppression technology application

According to quality control parameters, all HHP treatments resulted in significant downgrades of product quality. Also, the same treatments had no effect on suppressing the manifestation of soft-flesh associated with infestation of K. thyrsites. In addition to not controlling product quality, the implementation of this technology would lead to increased production costs as additional labour and facility upgrades would be required to conduct HHP on harvested fish upon arrival at the processing plant. For example, a limited volume of fish can be processed at a time, additional labour is required to load and unload the fish from the HHP machine (both would reduce the operational efficiency at the processing plant), in addition to added utility and maintenance costs for operating the machine. 

Recommendations for commercialization and implementation

The results of the pilot industrial trial demonstrate that HHP treatment proved not to be effective in controlling myoliquefaction, and resulted in significant deterioration in the quality of the product to levels that would be unacceptable for the consumer market. Therefore, HHP is not recommended for commercialization and implementation in the salmon processing industry.

Future Directions

Future steps should include the evaluation of other innovation applied technologies that could either suppress myoliquefaction or identify fish (screening) with high incidence of K. thyrsites infection prior to harvest. The salmon farm industry would have to prioritize which approach (suppression or screening) would be important to evaluate.

The BC Centre for Aquatic Health Sciences is interested in developing and/or testing non-invasive/non-lethal sampling technologies. Currently, one non-lethal and non-invasive screening technology that could be developed for monitoring K. thyrsites infection in live fish is Near-Infrared Spectroscopy. Preliminary tests have shown that Near-Infrared may be used as a non-lethal screening method for Bacterial Kidney Disease (BKD) (another important fish health issue affecting the aquaculture industry) in salmonids before development of external clinical signs.

An integrative approach involving different segments of the industry such as   salmon farmers, BC CAHS, and Near Infrared Spectroscopy technology providers would be necessary to obtain the resources to access this technology.

In order to build on this AIMAP Project, an important spin-off research and development project has recently been launched between the BC Centre for Aquatic Health Sciences and Marine Harvest Canada: Development of strategies to understand and mitigate the effects related to Kudoa thyrsites infection in Atlantic salmon (Salmo salar). The work plan of this 3-year project is designed to assess innovation technologies that will enable to determine when, where and how fish become infected with K. thyrsites. In addition, this project will assess non-lethal assay for diagnosing K. thyrsites. This initiative will be carried out at commercial culture conditions, which will enable a direct assessment of this fish health issue.

Acknowledgements

We wish to thank Tiffany MacWilliam (Marine Harvest Canada) for coordinating all aspects related to field sampling and testing, and laboratory analysis; Jeff Forbes (Co-op student from UVic at BC CAHS) and Debbie Collins (BC CAHS) for their support during field sampling and laboratory analysis. We wish to acknowledge that funds for this project (AIMAP Jan09-P25) were provided by the Aquaculture Innovation and Market Access Program.