Validation of a new commercial serine protease artificial digestion assay for the detection of Trichinella larvae in pork

Brad Scandrett,Patrik Buholzer,Alvin Gajadhar,Kelly Konecsni

doi:10.1016/j.fawpar.2018.04.001

Abstract

Trichinella is a zoonotic nematode parasite transmitted by the ingestion of raw or under-cooked meat. Control of the parasite is essential to facilitate public health and trade in products from susceptible food animals, including pork and horse meat. The standard method for detecting Trichinella muscle larvae uses pepsin enzyme and hydrochloric acid (HCl) in an artificial digestion procedure. A new artificial digestion assay using serine protease was recently developed and commercialized (PrioCHECK™ Trichinella AAD) for the detection of Trichinella larvae in the muscle of infected animals. The assay uses no hazardous substances such as HCl or pepsin. Activation of the enzyme requires an elevated digestion temperature of 60°C which kills the parasite and reduces the risk of contaminating the environment with Trichinella. Compared to the pepsin/HCl method, digestion time for the PrioCHECK Trichinella AAD assay is reduced by a third. A recent study demonstrated these features of the new assay and its suitability for digesting various muscles from domestic and wild animals. To further validate the assay's performance relative to the conventional pepsin/HCl digestion method several comparative studies were conducted using samples from different muscle sites spiked with low levels of encapsulated first stage Trichinella larvae (L1). Multiple muscle samples were collected from diaphragm, tongue, masseter, and loin of 3–4month old pigs. Samples were spiked with 3, 4, 5, or 25 Trichinella spiralis L1. A total of 320 meat samples of 100g each were used to compare the diagnostic proficiency of the PrioCHECK Trichinella AAD assay with the pepsin/HCl digestion method. Comparative and validation data produced from these studies showed that both methods are capable of consistently detecting Trichinella in 100g samples which contained as few as 3 L1 or 0.03 larvae per gram of meat. Overall, the PrioCHECK Trichinella AAD assay performed satisfactorily according to international guidelines of the World Organization for Animal Health (OIE), European Union (EU) and International Commission on Trichinellosis (ICT) for the detection of Trichinella infection in pork.

Highlights

Trichinella is a meat-borne nematode parasite that infects many species of animals
An additional clarification step was required in both methods to remove debris from the sediment that might obscure the detection of larvae during microscopic examination; this requirement was mostly for the PrioCHECK Trichinella AAD assay
Motile as well as non-motile larvae were recovered by the pepsin/hydrochloric acid (HCl) method whereas only non-motile L1 were recovered by the PrioCHECK Trichinella AAD assay

Summary

Introduction

Trichinella is a meat-borne nematode parasite that infects many species of animals. It is prevalent in wild carnivores and omnivores, with potential for spillover between susceptible species of wildlife and livestock (Pozio, 2015). There has been a close association between Trichinella spiralis and pigs, but other species of the parasite can infect porcine hosts Trichinella infection is not recognized as a cause of disease in animals, any species of the parasite can cause mild to serious illness in humans, and in some cases even death (Gottstein et al, 2009). A common tool for establishing or monitoring the effectiveness of control measures is the artificial digestion method which is used to detect first stage Trichinella larvae in muscle tissues of host species. In the EU alone, meat samples from millions of pigs are tested annually for the presence of Trichinella (Pozio, 2014)

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Conclusion