Larvae In Meat Research Articles

Inactivation of Trichinella spp. in naturally infected boar meat after Pulsed Electric Field (PEF) treatments

Larvae of the nematode Trichinella are capable of causing parasitic infections in humans after the consumption of uncooked meat or meat products, leading to severe symptoms and even death. Meat inspection is costly and tends to progressively be derogated in holdings applying controlled housing conditions in certain regions. Our study focuses on the evaluation of the efficacy of Pulsed Electric Fields (PEF) in the inactivation of Trichinella spp., including excysted larvae isolated by artificial digestion and encapsulated larvae found in meat from naturally infected wild boars. Microscopic examination of unstained and propidium-iodide-stained larvae suspensions showed that most of the excysted larvae were inactivated 10 min after an intermediate PEF treatment (1 kV/cm, 0.41 kJ/kg). Treating excysted larvae with the mildest PEF treatment (0.5 kV/cm, 0.05 kJ/kg) combined with a 3% NaCl incubation resulted in synergistic inactivation. The application of 3 kV/cm (20 kJ/kg) to wild boar meat resulted in the inactivation of over 90% of encapsulated Trichinella larvae. The viability of Trichinella in meat was inversely correlated to the field strength applied (1–6 kV/cm) for equal energy input (20 kJ/kg). These findings demonstrate that PEF technology can potentially serve as a novel strategy for the inactivation of Trichinella larvae in meat.

First use of tissue exudate serology to identify Toxocara spp. infection in food animals

Toxocara canis and Toxocara cati are globally distributed, zoonotic roundworm parasites. Human infection can have serious clinical consequences including blindness and brain disorders. In addition to ingesting environmental eggs, humans can become infected by eating infective larvae in raw or undercooked meat products. To date, no studies have assessed the prevalence of Toxocara spp. larvae in meat from animals consumed as food in the UK or assessed tissue exudates for the presence of anti-Toxocara antibodies. This study aimed to assess the potential risk to consumers eating meat products from animals infected with Toxocara spp. Tissue samples were obtained from 155 different food producing animals in the south, southwest and east of England, UK. Tissue samples (n = 226), either muscle or liver, were processed by artificial digestion followed by microscopic sediment evaluation for Toxocara spp. larvae, and tissue exudate samples (n = 141) were tested for the presence of anti-Toxocara antibodies using a commercial ELISA kit. A logistic regression model was used to compare anti-Toxocara antibody prevalence by host species, tissue type and source. While no larvae were found by microscopic examination after tissue digestion, the overall prevalence of anti-Toxocara antibodies in tissue exudates was 27.7%. By species, 35.3% of cattle (n = 34), 15.0% of sheep (n = 60), 54.6% of goats (n = 11) and 61.1% of pigs (n = 18) had anti-Toxocara antibodies. Logistic regression analysis found pigs were more likely to be positive for anti-Toxocara antibodies (odds ration (OR) = 2.89, P = 0.0786) compared with the other species sampled but only at a 10% significance level. The high prevalence of anti-Toxocara antibodies in tissue exudates suggests that exposure of food animals to this parasite is common in England. Tissue exudate serology on meat products within the human food chain could be applied in support of food safety and to identify practices that increase risks of foodborne transmission of zoonotic toxocariasis.

Trichinella proficiency testing in Southeastern European countries

In some Southeastern European (SEE) countries, like Serbia, Romania, and Bulgaria, trichinellosis is one of the most important foodborne zoonotic diseases. In those countries, EU regulation and local authorities require the staff of laboratories performing official controls on meat to be properly trained and to check their competence by participating regularly in proficiency testing (PT). PTs are organized by National Reference Laboratories for Trichinella of each country and involve all official laboratories testing meat. In Romania and Bulgaria, the organization of PT for the detection of Trichinella larvae in meat by Magnetic Stirrer Method (MSM) started in 2012. In Croatia and Serbia PT was first organized in 2015 and 2017, respectively. This study presents data on the performance obtained by official laboratories of SEE countries that organize PT at national level and compares the performance obtained by laboratories belonging to different countries. Results suggest that the constant participation in PT leads to an increase in the performance of participating laboratories by positively affecting the staff accuracy in sample testing by MSM. Since the percentage of recovered larvae was in some cases suboptimal (<80%) and occasionally very poor (<40%), there is room for improvement. The regular participation in PT by laboratories involved in official controls on meat intended for human consumption is fundamental to guarantee consumer safety.

Trichinella infection in Serbia from 2011 to 2020: a success story in the field of One Health.

In Serbia, modern pork production systems with implemented control measures, including the detection of Trichinella larvae in meat (ISO18743), have eliminated farmed pork from pigs slaughtered at abattoirs as a source of trichinellosis. Epidemiological data from 2011 to 2020 indicate that the number of human cases and the number of infected domestic pigs has decreased significantly. Over the years, pork was the most frequent source of human infection. Cases generally occurred in small family outbreaks, and the infection was linked to consumption of raw or undercooked pork from backyard pigs. In most of the outbreaks, T. spiralis was the aetiological agent of infection, but in 2016, a large outbreak was caused by consumption of uninspected wild boar meat containing T. britovi larvae. To achieve safe pork, it is important that consumers of pork from animals raised in backyard smallholdings and of wild game meat are properly educated about the risks associated with consumption of untested meat. Laboratories conducting Trichinella testing should have a functional quality assurance system to ensure competency of analysts and that accurate and repeatable results are achieved. Regular participation in proficiency testing is needed.

Efficacy of an aerosol-resistant pepsin powder used in artificial digestion for the detection of Trichinella larvae in meat.

Trichinellosis is an important worldwide foodborne zoonosis. The gold standard test to detect Trichinella spp. larvae in muscle samples of animals intended for human consumption is the artificial digestion method. Handling and dispensing of conventional pepsin powder present significant safety risks for analysts. The use of pepsin powder that is resistant to aerosolization should alleviate these safety concerns. The aim of this study was to compare the efficacy of an aerosol-resistant pepsin powder to conventional pepsin powder in the artificial digestion method. Proficiency samples of pork diaphragm containing specific numbers of viable Trichinella spiralis larvae were tested in two laboratories. The results revealed that aerosol-resistant pepsin was simple, effective and convenient to use, and showed good solubility and larval recovery that met the requirements of the European Union regulation EU 2015/1375. Overall, the efficacy of the aerosol-resistant pepsin was comparable to the conventional pepsin and safer for analysts.

Comparison of different techniques and stains for direct diagnosis of Trichinella spiralis

Background: The aim of Trichinella inspection is to reliably detect larvae in meat at levels which are capable of causing human trichinellosis. Trichinoscopy is a rapid, cost effective but is less sensitive in detection of low muscle larvae (ML)numbers. Artificial digestion is more convenient, and flexible, but may destroy immature larvae. Baermann technique may enhance the efficiency of diagnosis. Giemsa and Leishman stains provide identical contrasting coloration as Hematoxylin and Eosin (H-E) with the both advantages of that they can be used for staining non-histological sectionsand rapid preparation.The primary aim of the study was to compare the efficiency of digestion and Baermann techniques for detection of ML in muscles. Secondary aim was detection of ML by Geimsa and Leishman stain and their comparison with routinely used stain H-E. Methods: Muscles from 36 mice orally infected with Trichinella spiralis encysted larvae were examined. Larvae from 1 g samples of different muscles were recovered by digestion and Baermann techniques after different dose of infection. Muscle samples were prepared for staining with Giemsa, Leishman and H-E. Results: Number of larvae recovered by Baermann was higher recovered by digestion method (P ˃0.5). Leishman stain was the best stain for rapid detection of ML after 30 min followed in validity by Geimsa then H-E. Conclusion: Baermann concentration technique showed higher sensitivity than digestion method. Leishman stain is superior to Giemsa stain as it takes less time. Both stains are considered good alternative to H-E for rapid and easy diagnosis of trichinellosis in post-mortem animals.

Synthetic gene as target to assess the sensitivity of PCR to detect Trichinella spp. larvae in meat from a non-endemic region.

Trichinellosis is a zoonotic disease exotic in Brazil but commonly found worldwide including South American countries like Argentina. International trading of swine meat needs an official Trichinella-free diagnosis commonly carried out by pepsin-HCl digestion of diaphragm tissue fragments followed by microscopic examination for the presence or absence of Trichinella larvae. The easiness of this diagnostic method allows it to be performed at slaughtering plants but, in contrast, it lacks sensitivity and does not allow species differentiation, which is fundamental for determining geographical and species distribution of different genotypes. In our study, we aimed to evaluate a highly sensitive diagnostic method based on the polymerase chain reaction (PCR) that would allow us to detect and classify different species of Trichinella. Thus, we designed a synthetic gene and selected five sets of primers targeting specific regions of the Trichinella genome. The synthetic gene was cloned into a plasmid and then used to optimize PCR conditions. Using our PCR, we were able to detect 0.001pg of the synthetic gene, which corresponded to 0.01 larvae. Then, we collected 175 samples of Suidae (domestic and wild boars) diaphragm fragments that were pooled into groups, digested with pepsin-HCl, and had the DNA extracted for analysis by PCR. The clinical samples evaluated were negative by PCR. Our results indicate that the PCR-based method might be a useful diagnostic method complementary to the pepsin-HCl digestion method currently in use, mostly in non-endemic areas.

Epidemiological Studies on Porcine Trichinellosis in Five States of North East India

This study was carried out to evaluate the epidemiological studies of trichinellosis in five states of North East India from Apr 2016 to Dec 2017. Overall, 865 different meat samples for detection of Trichinella larvae and 1580 sera samples for detection of anti-Trichinella antibody were collected. Intensity of infection with Trichinella larvae in meat was determined by HCL: Pepsin digestion procedure and anti-Trichinella IgG in serum were detected using excretory/secretory antigens, according to validated ELISA. No Trichinella larva was detected by HCL: Pepsin digestion method. However, four (0.25%) samples were seropositive for Trichinella IgG and four inconclusive results as per cut off value. The highest seroprevalence was observed in Meghalaya (0.41%) followed by Assam (0.27%) whereas no seropositive cases were recorded in Arunachal Pradesh, Mizoram and Tripura. Trichinellosis is common in North East, India. However, it is suspected in communities where more than 75% of the population relish pork. Finally, there is a need for more research to establish the facts of trichinellosis in this region. Thus, public awareness, food hygiene, monitoring, and surveillance programme are suggested to implement for prevention of trichinellosis in this region.

First Serbian external quality assessment to detect Trichinella larvae in meat by the Magnetic Stirrer Method

Introduction. Inter-laboratory comparative testing must be conducted under the direction of the National Reference Laboratory for Trichinella in each Member State of the EU with the aim to ensure the quality of the approved digestion method and to evaluate the competence of laboratories in Trichinella detection. For the first time in Serbia, the National Reference Laboratory for Trichinellosis (NRLT INEP) organized such external quality assessment (EQA) for the detection of Trichinella larvae in meat by the Magnetic Stirrer Method (MSM). The aim of this study was to enable laboratories interested to take part in the EQA and to assess the quality of their MSM performance. Materials and Methods. The EQA was organized by NRLT INEP according to ISO/IEC 17043:2010. The EQA test panel, which was sent to eight laboratories within Veterinary Institutes, consisted of three 100?5 g minced pork meat balls with identical numbers of larvae. Two meat balls were spiked with five viable Trichinella spiralis muscle larvae (L1), and one meat ball was not spiked. Results and Conclusions. Evaluation made on the basis of qualitative results showed that 100% of participants successfully completed the testing. Quantitative evaluation showed that, on average, only 60% of Trichinella larvae were detected among all participants. The main importance of this study is that it enabled laboratories across the country to be compared. This should be efficient stimulus for improvement of analytical performance over time. Therefore, it will be of value if the participants and other official laboratories in Serbia take part annually in EQAs organized at national level. The results obtained in this study could provide useful information to the Ministry of Agriculture, Forestry and Water Management, Veterinary Directorate and be of importance for promoting the one health concept in the country as a whole.

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100 pg to 100 ag and found to be 10 fg, 1 fg and 100 fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.

Annual report of the Scientific Network on Microbiological Risk Assessment 2017

Annual report of the Scientific Network on Microbiological Risk Assessment 2017

Detection of Trichinella Larvae in Meat: Comparison of ISO 18743:2015 with Regulation (EU) 2015/1375

Trichinellosis is a foodborne parasitic zoonosis, caused by nematodes within the genus Trichinella. Pigs and wild animals are known sources of Trichinella infections for humans. The Commission Regulation 2075/2005 of the European Union (EU) established that all Trichinella spp. susceptible animals, which are suitable for human consumption, must be tested for these parasites using approved methodologies. Recently, the ISO 18743:2015 Standard on the detection of Trichinella larvae in animals was published. The purpose of this paper was to compare the method described in the ISO Standard with the reference method established in the Commission Regulation of the EU. The comparison was made by setting each clause of the standard versus the corresponding step of the Regulation. The artificial digestion of pooled muscle samples was recognized by both documents as the main reference method and the procedures described were very similar. The ISO Standard provides the basic principles and properties of the essential steps of the method and highlights the Critical Control Points (CCPs) of the procedure, which can affect the performance of the method, providing detailed instructions aimed at inexperienced analysts. The respective roles of the Standard and of the Regulation are also clarified. International standards are developed to provide agreed upon specifications relating to particular matters, while legislation sets mandatory requirements established by public authorities. Thus, European laboratories officially appointed for the detection of Trichinella in meat are required to apply the methods described in the Regulation, but may also rely on the ISO Standard for detailed steps and CCPs.

Proficiency testing to detect Trichinella larvae in meat in the European Union

According to the Commission Implementing Regulation (EU) 2015/1375 (replacing the Commission Regulation (EC) No 2075/2005), all animals, which are potential carriers of Trichinella spp. larvae, should be tested at the slaughterhouse or game-handling establishments according to one of the approved tests. One of the core duties of the European Union Reference Laboratory for Parasites is to organize proficiency testing (PT), as stated in the Commission Regulation (EC) No. 882/2004 of the European Parliament and of the Council. The aim of this work was to evaluate the results of PTs of the digestion method carried out by the National Reference Laboratories for Parasites (NRLPs) over a nine year period (2007–2015). Participating laboratories received a panel of samples consisting in 35g or 100g of minced pork or horse meat spiked with Trichinella spiralis live larvae. The number of spiked samples varied from 2 to 9 over the years. A negative control was also included in the panel, except during the 2015 PT, when only positive samples were used. The percentage of NRLPs, which passed the PT, increased from 83.3% in 2007 to 100% in 2014. Considering the number of recovered larvae, the heterogeneity in participant’s results reduced overtime. The values of the overall mean difference between spiked and recovered larvae decreased during the study period, witnessing a general improvement of NRLPs performance and confirming the effectiveness of PT for a good performance of this test.

The viability of Trichinella spiralis larvae in frozen pork and wild boar meat revealed by the experimental infection of laboratory mice

Summary The aim of the research was to verify the viability of Trichinella spiralis larvae in pig and wild boar meat frozen at -18 °C from 268 days to 1021 days. There were three groups of laboratory inbred Balb/C mice and Bagg/ Albino/ strain C, each group received one sample consisting of 20 g meat infected with T. spiralis larvae, according to the protocol, and an uninfected control group. The experiments were ended after 80 days, by euthanizing the mice and taking samples of muscle tissue, which were processed for histopathology. The results revealed the presence of Trichinella cysts in all experimental groups, confirming the extraordinary durability and viability of Trichinella larvae in meat frozen at -18 °C for many months. It was confirmed that the larvae detected in muscle tissues of mice were Trichinella spiralis.

Sensitivity and Optimization of Artificial Digestion in the Inspection of Meat for Trichinella spiralis

In many countries, the method of choice in inspecting meat for Trichinella spiralis infection is artificial digestion. We conducted a study of the sensitivity of the artificial digestion method recommended by the International Commission on Trichinellosis for detecting T. spiralis larvae in meat and of the effect of modifications of some procedures used in the method on its sensitivity. As part of this, we evaluated the effects on larval recovery of the vessels used for larval settling, sieve sizes, and temperatures at which larvae passed through the sieves, using larvae from T. spiralis-infected mice. We observed the effects on larval recovery of digestion duration and of modified artificial digestion by using 10-g samples of infected mouse muscle alone or mixed with uninfected pork. The percentages of larvae recovered with the respective use of separatory funnels and conical cylinders were 51.20% and 98.70%. The rates of recovery of T. spiralis larvae at 4 degrees C after passage through sieves of 425-microm mesh (No. 40), 250-microm mesh (No. 60), and 180-microm mesh (No. 80) were 98.42%, 90.59%, and 81.63%, which exceeded the 97.79%, 85.10%, and 61.12% rates of recovery of motile larvae at 40 degrees C and the 95.12%, 78.60%, and 44.16% rates of recovery of dead larvae at 90 degrees C. The larval recovery rate after digestion for 2 hours (96.18%) was greater than that after 0.5 hours (88.00%). We then examined a modified digestion method in which 10-g samples of pork mixed with 300 mL of digestive solution were digested for 2 hours at 43 degrees C followed by chilling of digest solution to 4 degrees C before passing it through a 425-microm mesh (No. 40) sieve and allowing it to settle in a 1-L conical cylinder. With this procedure, the modified method detected T. spiralis in samples of pork meat weighing 10 g and containing either 1 larva per gram or 0.1 larva per gram. Further validation of digestion method incorporating these modifications is required with the use of larger samples of infected muscle from species such as swine, which are routinely tested for T. spiralis for the purpose of food safety.

Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR

Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA ( rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C T values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.

Trichinella spiralis: Effect of high temperature on infectivity in pork

Twenty gram samples of homogenized Boston shoulder from swine experimentally infected with Trichinella spiralis were sealed in plastic pouches, pressed to a uniform thickness of 2 mm, and subjected to water bath temperatures of 49, 52, 55, 60, and 63 ± 0.5 C for intervals of 2 min to 6 hr, especially within the interval of 0 to 15 min. These times included a period of about 1 min at the start and a period of about 1 min at the end for temperature equilibration. Treated samples were rapidly chilled to 25 C and then digested in a 1% pepsin-HCl solution at 37 C for 18 hr to recover T. spiralis larvae. The recovered larvae were suspended in 2 ml saline; 1 ml of this suspension was introduced into the stomach of each of two rats. The linear equation, log (time) = 17.3 − 0.302 (temperature), was calculated from the time required at each temperature for the inactivation of T. spiralis larvae. The correlation coefficient for that relationship was r = −0.994. Larvae heated in the meat to 55 C for 4 min retained their infectivity, but were rendered noninfective after 6 min at 55 C. At 60 C, larvae were not infective after only 2 min (zero dwell time); whereas at 52 C, 47 min were required to render the larvae noninfective. Larvae in meat heated to 49 C were infective after 5 hr but not after 6 hr. These data demonstrate that the destruction of infectivity of T. spiralis is time-temperature related.