Trend analysis of Trichinella in a red fox population from a low endemic area using a validated artificial digestion and sequential sieving technique.

Frits Franssen,Arie Havelaar,Zanda Esíte,Joke Van Der Giessen,Arno Swart,Gunita Deksne

doi:10.1186/s13567-014-0120-9

Abstract

Freezing of fox carcasses to minimize professional hazard of infection with Echinococcus multilocularis is recommended in endemic areas, but this could influence the detection of Trichinella larvae in the same host species. A method based on artificial digestion of frozen fox muscle, combined with larva isolation by a sequential sieving method (SSM), was validated using naturally infected foxes from Latvia. The validated SSM was used to detect dead Trichinella muscle larvae (ML) in frozen muscle samples of 369 red foxes from the Netherlands, of which one fox was positive (0.067 larvae per gram). This result was compared with historical Trichinella findings in Dutch red foxes. Molecular analysis using 5S PCR showed that both T. britovi and T. nativa were present in the Latvian foxes, without mixed infections. Of 96 non-frozen T. britovi ML, 94% was successfully sequenced, whereas this was the case for only 8.3% of 72 frozen T. britovi ML. The single Trichinella sp. larva that was recovered from the positive Dutch fox did not yield PCR product, probably due to severe freeze-damage. In conclusion, the SSM presented in this study is a fast and effective method to detect dead Trichinella larvae in frozen meat. We showed that the Trichinella prevalence in Dutch red fox was 0.27% (95% CI 0.065-1.5%), in contrast to 3.9% in the same study area fifteen years ago. Moreover, this study demonstrated that the efficacy of 5S PCR for identification of Trichinella britovi single larvae from frozen meat is not more than 8.3%.

Highlights

Trichinella species infect a wide range of mammals, including humans [1,2]
We show that the recovery rate of spiked dead Trichinella larvae in meat digest is 60% using EU-RM, while the recovery rate using sequential sieving method (SSM) is 92%, making SSM the technique of choice to detect dead Trichinella larvae in frozen meat
Validation of larva detection The sensitivity to detect dead Trichinella muscle larvae (ML) in meat digest of the EU-RM was 60% (n = 100), whereas the SSM performed significantly better with 92% (n = 100) sensitivity (p = 6 · 10-12, Fisher’s Exact test) (Table 1)

Summary

Introduction

In the European Union, the magnetic stirrer method (EU reference method, EU-RM) according to European regulation EC 2075/2005 [3] is used for individual carcass control of Trichinella susceptible animals intended for human consumption and for surveillance of Trichinella infections in wildlife. This method includes two consecutive sedimentation steps to isolate Trichinella muscle larvae (ML) and has been validated for the detection of live larvae, for which critical control points are well described [4]. Dyer and Evje [20] recovered only 80% of spiked dead Trichinella ML in 2 litres Trichinellafree meat digest after one hour of sedimentation (twice the time routinely used in EU-RM)

Methods

Results

Conclusion