Pooled Sample Digestion Research Articles

Validation Parameters of the Magnetic Stirrer Method for Pooled Sample Digestion for Trichinella spp. in Horse Meat Based on Proficiency Tests Results.

Meat of horses may be infested with nematodes of the genus Trichinella spp. and can cause serious disease in humans. Rules for the carcasses sampling of species susceptible to Trichinella spp. infection and examination are laid down in Commission Regulation 1375/2015, where the magnetic stirrer method for pooled-sample digestion is recommended (Commission Regulation 1478/2020). All personnel involved in the examination should be properly trained and participate in quality control programs. Proficiency tests (PTs) play a key role in the quality verification process. This paper presents the results of PTs organized for 68 Polish laboratories in 2014-2019. Results were assessed qualitatively at three levels of sample contamination (0, 3, 5 larvae) and quantitatively at one level (5 larvae). The laboratories have achieved the average correct qualitative results 100%, 96.2% and 96.8% for the samples contaminated with 0, 3 and 5 larvae, respectively. In the quantitative evaluation, an average 94.1% of the reported results were correct. The data from PTs enabled us to define, for the first time, validation parameters of the digestion method for the horse meat matrix in a large-scale experiment including: specificity (100%), sensitivity (95.6%), accuracy (97.1%), the limit of detection (LOD) (1.14 ≈ 1) and the limit of quantification (LOQ) (3.42 ≈ 3).

Distribution of Trichinella spiralis, Trichinella britovi, and Trichinella pseudospiralis in the Diaphragms and T. spiralis and T. britovi in the Tongues of Experimentally Infected Pigs.

There is little or even no data in the global literature on the distribution of different species of Trichinella in the individual parts of the diaphragms and tongues in infected pigs. This is of particular importance from the food safety point of view and for the conduct of routine testing of pig carcasses for Trichinella as well as epidemiological surveys. Therefore, the aim of the present study was to evaluate the distribution of Trichinella spiralis (T. spiralis), Trichinella britovi (T. britovi), and Trichinella pseudospiralis (T. pseudospiralis) ML in various parts of the diaphragm (the pillars, costal, and sternal part) and the distribution of encapsulated species of Trichinella (T. spiralis and T. britovi) in various parts of the tongues (the tip, body, and root) of experimentally infected pigs. The diaphragm pillars were the most heavily parasitized part of the diaphragm both in groups of pigs infected with particular species of Trichinella and in groups of pigs presenting different levels of infection; however, statistical differences were observed only in the group of pigs with moderate (21–35 larvae per gram-lpg) or moderately high (35–55 lpg) intensity of Trichinella spp. infection in the entire diaphragm. In all groups of pigs, regardless of the infecting Trichinella species or infection level, larvae showed a homogeneous distribution on both sides of the diaphragm and excluding those of T. pseudospiralis, also in all three parts of the tongue. Histological examination showed features of a differential inflammatory response around larvae of the different Trichinella species. This study confirmed that for mandatory examination of pig carcasses using a pooled-sample digestion assay in which each pig is intended to be represented by a 1 gram sample taken from the diaphragm pillars, if that tissue is not available, the mass of the sample taken from the remaining diaphragm parts (costal or sternal) should be at least double that from the pillars. Histological findings confirmed that the inflammatory pattern of pig muscles varies depending on the Trichinella species triggering the infection and is less intense in the case of infections with T. pseudospiralis than in infections with encapsulated species of Trichinella (T. spiralis and T. britovi).

Test sensitivity of a commercial serine protease digestion kit for the detection of Trichinella spiralis and Trichinella pseudospiralis larvae in pig muscle

The reference method for Trichinella detection at meat inspection is the magnetic stirrer method (MSM) utilising HCl-pepsin for pooled sample digestion. Due to availability and quality issues with pepsin, alternative digestion methods are being offered, such as the Priocheck Trichinella AAD kit (T-AAD), based on serine endopeptidase digestion. In this study the T-AAD kit was compared to the reference method.Minced pork samples were spiked with T. spiralis muscle larvae (ML) with- and without capsule or T. pseudospiralis ML, and analysed with both tests. Test results of individually spiked test samples were analysed by generalised linear modelling.The T-AAD test kit was comparable to the reference method for the qualitative detection of T. spiralis in pigs, but not quantitatively. Overall, 94% of spiked T. spiralis were recovered using MSM against 75.2% when using T-AAD (p < 0.0001). Using the MSM 80.0% of spiked T. pseudospiralis were recovered against 20% with the T-AAD (p < 0.0001).Based on our experience with the T-AAD kit, we strongly recommend validating the method on site prior to introduction into routine diagnostic laboratories, but this will not alleviate the poor test sensitivity of the T-AAD for the detection of T. pseudospiralis.

Prevalence of Trichinella britovi in muscle tissue samples from foxes from Podkarpackie Voivodeship, Poland

The aim of the study was to detect Trichinella larvae in muscle tissue samples of red foxes (Vulpes vulpes) living in Podkarpackie Voivodeship and to determine their species composition. Muscle tissue samples had been collected from a total number of 139 red foxes culled during 2011-2012 in Podkarpackie Voivodeship and studied for the presence of Trichinella. Ten-gram muscle tissue samples were collected from each animal, combined into a fifty-gram pooled sample and then tested for the presence of Trichinella by the reference method for pooled sample digestion with a magnetic stirrer. Whenever a pooled sample gave a positive result, the suspected animals were resampled, and each ten-gram muscle tissue sample was tested separately. Trichinella larvae were identified to species by Multiplex PCR. The studies revealed the presence of one Trichinella larva in a pooled sample composed of five individual samples from foxes culled in the following locations: Leszczowate in Bieszczadzki District (one sample), Szczutków in Lubaczowski District (two samples), Podemszczyzna in Lubaczowski District (one sample) and Strzyżów in Strzyżowski District (one sample). No Trichinella larvae were detected during the test carried out on individual samples that composed the pooled sample. Numerous Trichinella larvae were found in samples from 4 foxes coming from Berezka and Glinka in Leski District, Dybawka in Przemyski District, and Golcowa in Brzozowski District. All Trichinella larvae isolated from the foxes belonged to the species T. britovi. The study confirmed the role of foxes as an important vector of Trichinella larvae circulating in the habitat of humans and animals. T. britovi is a species occurring most frequently in the fox population.

Detecting Trichinella infections using inverse microscopy and an improved larval counting technique

Several methods for the detection of Trichinella in meat are legally prescribed in regulation (EC) No 2075/2005, which prescribes the magnetic stirrer method for pooled sample digestion (MSM) as the reference method. However, the MSM's multistage protocol requires several preparatory steps that seem to be accountable for the loss of larvae. Here we present a modified MSM (mMSM) based on: (1) an inversion of the optical path using inverse microscopy; and (2) a modified larval counting basin (mLCB, 'Trichoview'). This enables one to examine samples of up to 40ml and reduces the examination area from 72 to 10.3cm2. Preparatory steps that might cause the loss of Trichinella larvae are eliminated from the new protocol. Correspondingly, the overall analytical time is reduced. In a direct and blinded comparison using 60 digest samples containing spiked vital Trichinella larvae (1-90 L1), both methods performed well for both small and large numbers of L1. However, 1278 of 1285 L1 (99.4%) were detected using the mMSM, while MSM recovered only 1225 L1 (95.3%). The improvement stems largely from samples with small numbers of L1: in all samples spiked with fewer than 10 L1, the recovery rate of mMSM was 100% compared to only 93% with MSM. Our data suggest that the use of the mMSM can improve the recovery rate by about 4% and therefore reduce the chances of a false-negative result in a sample containing 5 larvae by a factor of about 4.

Trichinella detection: Identification and statistical evaluation of sources of error in the magnetic stirrer method for pooled sample digestion

Proficiency testing (PT) is the use of inter-laboratory comparisons to determine the performance of individual laboratories for specific tests or measurements, and to monitor a laboratory's performance. Participation in proficiency testing provides laboratories with an objective means of assessing and demonstrating the reliability of the data they are producing. To ensure the reliability of Trichinella detection and meat hygiene within the European Union and afford optimal protection to the consumer, PT is conducted under the direction of the European National Reference Laboratories for Trichinella. Evaluation of data from the national PT showed that lab-internal shortcomings are frequent. These shortcomings are specifically related to: (1) improper sample collection and preparation; (2) incorrect transposition and application of the protocol as laid down in Annex I, Chapter I, Nr. 3 (a–g) of the Commission Regulation (EC) No. 2075/2005; (3) insufficient sedimentation times; and (4) improper equipment.(e.g. Prost and Nowakowski, 1990; Rossi and Pozio, 2008; Forbes and Gajadhar, 1999; Rossi and Pozio, 2008). To test the hypothesis that both method based errors as well as internal lab errors can influence the accuracy and precision of the magnetic stirrer method for pooled sample digestion (MSM), we initiated a study to evaluate the analytical uncertainty of the MSM. Results presented here are based on: (i) data from PT in Germany (2008, 2009, and 2010); (ii) within-lab performance conducting high volumes of MSM; (iii) larval recovery experiments; and (iv) statistical evaluation of data resulting from these procedures. Quantitative data from the PT show that on average only 60% of Trichinella larvae were detected. Even laboratories that showed relatively good performance (>80% larva recovery, no false negative or false positive results), frequently reported samples with an unexpectedly low larval count (loss of >2 larvae). In our own laboratory, high numbers of repeated analyses of standards and re-analyses of residual fluids indicated that these outliers could be described by a binomial distribution based on a laboratory-specific Trichinella-detection probability. Results of recovery experiments indicate that only a part of the total larval losses can be attributed to lab-internal shortcomings inasmuch as a significant number of L1 could be isolated from the residual and washing fluids.

A novel detection method for Alaria alata mesocercariae in meat

Distomum musculorum suis (DMS), the mesocercarial stage of the trematode Alaria alata, can cause severe damages within their hosts, and since several reports about cases of human larval alariosis have been published, it became apparent that infected game animals and in particular wild boars are a potential source of infection for both humans and animals. A final statement concerning the health risks for consumers could not be given due to the lack of information about both the prevalence of DMS and the suitability of Trichinella inspection methods to detect this parasite in wild boar meat. Our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for DMS detection in wild boars, (2) development, optimization, and validation of methods, and (3) the distribution of the parasites within their paratenic hosts. A total of 868 individual samples/digests from 48 wild boars were analyzed by the reference method for Trichinella detection in meat samples according to regulation (EC) No. 2075/2005. In addition to the official protocol, a method modification with Pankreatin(c) and bile acid was applied for analysis of adipose tissue samples (n = 89). On the basis of our results, a new detection method based on a larvae migration technique was developed and used for detection of DMS in 574 single samples. Furthermore, the distribution patterns of DMS in wild boars in a total of 1377 single sample migrations/digestions from 35 positive animals were analyzed by application of all three methods. The official digestion method for Trichinella spp. in wild boars meat is inapplicable for the detection of A. alata mesocercariae as it shows shortcomings in both digestion and sampling. A direct comparison between the newly developed A. alata mesocercariae migration technique and the official digestion method for Trichinella spp. based on 574 single samples from 18 animals clearly shows that the sensitivity to detect A. alata developmental stages in tissues of wild boars of the new method is nearly 60% higher compared with the magnetic stirrer method for pooled sample digestion as laid down in regulation (EC) No. 2075/2005. Among other advantages, this method offers a simple, highly applicable, fast, and cost effective way to detect DMS in wild boars which is already applicable in routine veterinary inspection.

Control of trichinellosis by inspection and farm management practices

The prevention of human trichinellosis by proper meat inspection is a classic example of successful veterinary public health measures. The microscopic methods which have been used for more than a century to test pigs for trichinae were intended to prevent human disease. However, the value of these relatively insensitive direct detection methods, including trichinoscopy and pooled sample digestion, was debated as soon as more sensitive indirect (serological) methods became available. Two issues related to testing were discussed. First, should public health authorities endeavour to prevent all infections of humans rather than simply prevent the occurrence of disease, and second, would epidemiological surveillance and monitoring of the pig population on farms not provide a better control system to prevent human infection. This latter issue is of particular importance for those countries in the world where human trichinellosis acquired from farmed animals is absent and examination of pigs at the abattoir only results in negative findings. In countries where domestic pig infections are virtually non-existent, monitoring of Trichinella infection in wildlife could also contribute to understanding the infection pressure from nature to livestock. Trichinella-free pig farming is a feasible option for controlling this zoonosis, even in endemic areas. This approach provides an opportunity to combine good veterinary practice, in order to prevent animal diseases, with the prevention of Trichinella infection. All animals with access to the environment, or animals which are fed with potentially Trichinella-infected feed (swill, carcasses) will always constitute a public health threat, and must be inspected individually at slaughter (swine, horses, wild boars). Finally, it is important to recognize that trichinellosis is a world-wide problem that needs continuous public health attention. If no control system exists, for whatever reason, the public should be educated not to consume improperly cooked meat.

Factors affecting the efficiency of pooled sample digestion for the recovery of Trichinella spiralis from muscle tissue

Inspection for Trichinella spiralis in pork, horse and game meats is an important part of veterinary public health programmes in many countries. Variations of the pooled sample digestion test are the most widely used methods of inspection for this parasite. In this study, several aspects of the test, including sample preparation, reagent concentration and sample processing were examined for effect on test efficiency. Current methods using sample blending were equivalent or superior to sample grinding. Increasing the concentration of pepsin improved overall digestion slightly. The most critical factor affecting parasite recovery was sieve size. Currently used methods that employ a #80 (180 μm mesh) sieve limit the recovery of motile or dead parasites and therefore might decrease test sensitivity. It is recommended that a #45 (355 μm mesh) sieve be used to ensure optimal recovery of larvae.

Detection of Trichinellosis in Pigs by Artificial Digestion and Enzyme Immunoassay

The objective of this study was to demonstrate the reliability of current and proposed methods for the inspection of swine and other species for infection with the parasite Trichinella spiralis.Five groups of pigs were infected with doses of 2500, 500, 100, 50, and 20 T. spiralis larvae to establish moderate and low-level infections. Pigs were bled periodically during the study for samples to be tested by enzyme immunoassay (EIA). At the conclusion of the study, pigs were slaughtered and tissues collected for analyses of worm burdens and for comparison of digestion testing methods. Comparisons of pooled sample digestion methods were made using inspection methods prescribed by European Union Directives and the USDA, Code of Federal Regulations.Pooled sample digestion testing using 1-g samples was effective for detecting pigs with larval densities of >10 larvae per gram (LPG) of tissue but only partially effective for pigs with infections of <3 LPG. Pooled sample digestion testing using 5-g samples detected all pigs with infection levels >1 LPG. The EIA detected all T. spiralis-infected pigs, but did not detect infections in some pigs until 49 days after inoculation. These results demonstrate that the pooled sample digestion method using a 5-g sample size is the most effective inspection method for reducing the risk of human exposure to T. spiralis in pork.

Husbandry, parasitic and other diseases as factors in the reliability of the Enzyme Linked Immunosorbent Assay (ELISA) for detection of trichinellosis in pigs

Serum samples were obtained from pigs originating from specified pathogen-free farms, large industrialised farms and small conventional farms. All animals proved to be free of Trichinella spiralis by a pooled sample digestion method. Careful meat inspection studies on parasitic infections other than trichinellosis, and other inflammatory reactions were recorded and used for subdivision of the animals in different groups. It was concluded that animal husbandry, parasitic infections, or inflammatory reactions have not influenced the mean extinction values of an ELISA for the detection of T. spiralis antibodies, however, the specified pathogen free animals yielded somewhat lower results.

Comparison of three methods for detection of prolonged experimental trichinellosis in pigs

Three methods were employed for the diagnosis of porcine trichinellosis. The pooled sample digestion method and trichinoscopy served as European Community (EC) reference techniques, whereas the reliability of the Enzyme Linked Immunosorbent Assay (ELISA) was tested by 11 laboratories of the European Community and Sweden. Three groups of 6 piglets each were orally inoculated with 50, 150 and 1500 Trichinella spiralis larvae into each animal. Another group of 6 animals served as a non-infected control. Animals were slaughtered and serum and muscle samples were collected at Weeks 4, 12 and 40. The material was mailed under code and examined in all participating laboratories. ELISA proved to be a sensitive technique. ELISA micro assay was the most sensitive procedure. Of the direct techniques the reference pooled sample digestion method was more sensitive than trichinoscopy. It was concluded that both micro and macro ELISA can be used with confidence for the detection of low grade, longstanding experimental T. spiralis infections in swine.