A Validated Trichinella Digestion Assay and an Associated Sampling and Quality Assurance System for Use in Testing Pork and Horse Meat

Abstract

A revised digestion method, developed for efficiency and quality assurance, was validated for the detection of Trichinella larvae in pork and horse meat to meet requirements for food safety testing and facilitate access to international markets. The method consisted of a tissue homogenization step and a spin bar digestion procedure conducted at 45 degrees C to free larvae from muscle tissue, followed by two sequential separatory funnel steps to concentrate the larvae for detection using a stereomicroscope. Critical control points were determined for the method and monitored during testing. Under conditions of a defined protocol, test capacity was suitable for industrial applications, since multiples of up to 100 g of tissue could be analyzed at one time. The overall sensitivity of the test system depended on the size and origin of the sample taken from individual infected carcasses. Data from swine indicated that the currently accepted sample size of 1 g from individual carcasses consistently detected larval loads of > or =3 larvae per gram. Larval loads of 1.0 to 1.9 larvae per gram required 3- to 5-g samples of muscle tissue for reliable detection. Five-gram samples were considered optimal, because they consistently detected more tissues than 3-g samples, although the difference was not statistically significant. Tissue localization studies in experimental pigs indicated that the tongue and diaphragm were the tissues of choice for the most sensitive larval recovery. A system of analyst training, laboratory certification based on ISO guide 25, and on-site proficiency panel testing was used to ensure that external laboratories would consistently produce reliable test results. The system developed for pork was successfully modified for the testing of horse meat.

Comparing the demonstration of freedom fromTrichinellainfection of domestic pigs by traditional and risk-based surveillance

“…Serology has two advantages over the routine artificial digestion test. First, especially with low larval densities the diagnostic sensitivity of ELISA and WB is higher than of routine artificial digestion [10,20,[25][26][27][28]. Second, the number of larvae triggering a detectable antibody response is much lower than the number of larvae that can be detected reliably by routine artificial digestion test [29], leading to a higher analytical sensitivity of serology.…”

“…The sensitivity of this method depends on the larval density of the positive samples. Above a larval density of 3-5 larvae per gram (LPG), a sensitivity of 100 % was achieved, but below 1 LPG the sensitivity dropped to 40% [10]. Because y15-20 % of naturally infected pigs harboured larval densities of <1 LPG [11], infected pigs may not be detected reliably by this method.…”

Detection of Experimental Swine Trichinellosis Using Commercial Elisa Test

“…Our findings indicate that the highest intensity of T. spiralis infection occurred in the diaphragm. A large number of authors have identified swine diaphragm and tongue as equally valuable research material for T. spiralis muscle larvae detection (Kotula et al 1984, Gamble 1996, Forbes and Gajadhar 1999, Serrano et al 1999. In the present study T. spiralis infection intensity averaged to 24.30 larvae/g in the tongue, which was significantly lower (53%) than in the diaphragm (46.18 larvae/g = 100%).…”

Evaluation of a New Commercial Enzyme-Linked Immunosorbent Assay for the Detection of Porcine Antibodies against Trichinella Spp.

“…8 In contrast, artificial digestion using 1-g samples can only reliably detect positive samples above the level of 3-5 LPG. 6,22 In the current evaluation study, the commercial ELISA was correctly able to detect antibodies in samples from pigs with larval densities of 0.025 LPG, which was the lowest larval load in this sample set. However, 2 samples with larval burdens of 0.4 and 32 LPG were not detected in the commercial ELISA but also tested negative in the in-house ELISA, the in-house Western blot, or both.…”

“…The sample size had to be increased to 3-5 g to detect samples containing a minimum of 1-2 LPG. 6,22 The minimum infectious dose for humans was earlier estimated at 60-750 infective larvae (European Food Safety Authority: 2005, Risk assessment of a revised inspection of slaughter animals in areas with low prevalence of Trichinella. Available at http://www.efsa.europa.eu/cs/BlobServer/ Scientific_Opinion/biohaz-ej200-op-trichinella-en%20vf.…”