Methods for investigating Trichinella infections in domestic and wild animals

Trichinellosis is an important parasitic zoonosis that is caused by the intracellular nematode Trichinella spp.. Infection of humans occurs through consumption of raw (or undercooked) meat containing infectious larvae. In Europe, meat from pork, horse, and wild boar have been identified as most important sources of Trichinella infections in humans. In Switzerland, both the domestic pig and wild boar population are considered free of Trichinella. Conversely, Swiss foxes, lynxs and recently a wolf were found to be infected, the species identified in these animals was always referred to as Trichinella britovi. Although this species rarely infects pork and, compared to Trichinella spiralis, only causes reduced pathogenic effects in humans, the basic presence of Trichinella in Switzerland cannot be neglegted. This fact has gained increasing importance since the responsible authorities in the European Union (EU) are preparing regulations for the official Trichinella-control in meat in order to improve food safety for consumers. These regulations will be implemented as a consequence of the recent association of east European countries with the EU. This new legislation particularly takes into account, that in the past by far most cases of human trichinellosis in the EU were due to consumption of imported east European meat.Within the framework of the bilateral agreements of Switzerland with the EU, the Swiss veterinary public health authorities will have to comply with the foreseen EU regulations. Although diagnostic methods for the direct demonstation of Trichinella in pork meat are already routine practice in several Swiss abattoirs, the implementation of a meat control program for Trichinella for the entire slaughter pig population of the country would lead to an enormous increase in costs for the administration and will require an increased infrastructure in veterinary services. In order to find a reduced testing format for monitoring Trichinella infections in Swiss pork, an infection risk-oriented survey strategy is currently evaluated. In the present article, this minimized survey strategy is discussed regarding its compatibility with the EU regulations laying down rules for the official control of meat for Trichinella.     

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.     

Current status of the discussion on the certification of so-called "Trichinella-free areas"

In the last century already, Friedrich Albert Zenker and Rudolf Virchow have provided the essential prerequisites for the examination of pork for Trichinella in Germany. According to Directives 64/433/EEC and 77/96/EEC, this examination is still required in all member states of the European Union (EU) for trade between EU countries and for pork imported from third countries. According to Directive 64/433/EEC (Article 6, para 2), the examination of swine for Trichinella may be dispensed with if, on the basis of epidemiological studies, the stocks are found to be free from Trichinella and there is an efficient system of ante-mortem and post-mortem examination and control. However, it had been impossible to introduce these provisions into practice due to the fact that criteria were lacking. Therefore, the Scientific Veterinary Committee of the European Commission established a working group which has elaborated a concept for the certification of Trichinella-free pig stocks. The concept includes, inter alia, definitions of the essential components of "non-endemic area" and "Trichinella-free stock" and the requirement to be complied with for certification. Details are given on measures taken to control continued compliance with the certification requirements, the reporting and documentation system and the measures to be taken when trichinellosis is detected at a certified farm.     

Detection and surveillance for animal trichinellosis in GB

The zoonotic disease trichinellosis is considered one of the re-emerging diseases with surveillance and control methods constantly gaining more importance worldwide. Recent change in European Union (EU) legislation introduces Trichinella-free production, and the possibility of risk-based monitoring for Trichinella in pigs. This has increased the role of wildlife surveillance programmes and their impact on protecting human health as well as highlighted the need for harmonised surveillance protocols and test methods for these infections. A modified digest method, based on the EU reference method for Trichinella testing of pig meat, was used to screen foxes present in Great Britain (England, Scotland and Wales) for trichinellosis. The method was validated using batched pools of 10 g foreleg muscle from up to 20 foxes (maximum amount 200 g). The method gave an average trichinae recovery rate of 71% for spiked samples. Assuming this recovery rate applies to all contaminated samples, then the test sensitivity would be 70% for all tissue samples with 0.1 trichinae per 10 g of foreleg muscle, 99.9% for samples with 1 trichinae per 10 g, and 100% for samples with 2 or more trichinae per 10 g. In two separate studies, conducted between 1999 to 2001 (Smith et al., 2003) and 2003 to 2007, over 3500 wild foxes have been screened for Trichinella with negative results. In the second study reported here, foxes were collected from locations throughout Great Britain using a stratified sampling method based on fox population densities. All work was conducted in compliance with appropriate quality assurance systems, latterly under ISO 9001. Results to date indicate the national prevalence of trichinellosis in foxes is <0.001 based on a 10 g individual sample size, an infection level of 1 larva per gram (l pg) and 95% confidence interval. This, together with no reports of trichinellosis in domesticated pigs, suggests that Britain can be considered a region of negligible risk of trichinellosis.     

Human outbreak of trichinellosis in the Mediterranean island of Sardinia, Italy

Trichinella sp. infection has been documented in both humans and animals in most Mediterranean countries, yet in the past 60 years no infections have been reported on the Mediterranean islands. We describe the first outbreak of Trichinella sp. infection to have been reported on the island of Sardinia. The outbreak occurred in two villages in 2005 and involved 11 persons who had eaten raw sausages made from the same free-ranging sow. All 11 persons developed signs and symptoms of trichinellosis and seroconverted within 48 days of consuming the infected meat. The etiological agent was Trichinella britovi. Sardinia, like all Mediterranean islands, had been considered to be Trichinella-free. The present report, together with a recent report of T. britovi infection in animals on the nearby island of Corsica (France), raises questions as to the validity of the concept of Trichinella-free areas or regions.     

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.     

Good animal welfare makes economic sense: potential of pig abattoir meat inspection as a welfare surveillance tool

ABSTRACT: During abattoir meat inspection pig carcasses are partially or fully condemned upon detection of disease that poses a risk to public health or welfare conditions that cause animal suffering e.g. fractures. This incurs direct financial losses to producers and processors. Other health and welfare-related conditions may not result in condemnation but can necessitate 'trimming' of the carcass e.g. bruising, and result in financial losses to the processor. Since animal health is a component of animal welfare these represent a clear link between suboptimal pig welfare and financial losses to the pig industry.Meat inspection data can be used to inform herd health programmes, thereby reducing the risk of injury and disease and improving production efficiency. Furthermore, meat inspection has the potential to contribute to surveillance of animal welfare. Such data could contribute to reduced losses to producers and processors through lower rates of carcass condemnations, trimming and downgrading in conjunction with higher pig welfare standards on farm. Currently meat inspection data are under-utilised in the EU, even as a means of informing herd health programmes. This includes the island of Ireland but particularly the Republic.This review describes the current situation with regard to meat inspection regulation, method, data capture and utilisation across the EU, with special reference to the island of Ireland. It also describes the financial losses arising from poor animal welfare (and health) on farms. This review seeks to contribute to efforts to evaluate the role of meat inspection as a surveillance tool for animal welfare on-farm, using pigs as a case example.     

Larval viability and serological response in horses with long-term Trichinella spiralis infection

The horse is considered an aberrant host for the nematode parasite Trichinella spiralis, and many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood. It has been reported that experimentally-infected horses produce a transient serological response to infection and that muscle larvae are cleared more rapidly than in parasite-adapted hosts such as the pig and humans. However, limited numbers of animals have been studied, and both the longevity of larvae in horse musculature and the immune response to Trichinella larvae remain unclear. In this study, we infected 35 horses with 1000, 5000, or 10,000 T. spiralis muscle larvae and followed the course of infection for 1 year, assessing larval burdens in selected muscles, the condition and infectivity of recovered larvae, and the serological response of infected horses. The results demonstrated that T. spiralis establishes infection in horses in a dose dependent manner. Anti-Trichinella IgG antibodies peaked between weeks 6-10 post-inoculation. Viable, infective larvae persisted in horse musculature for the duration of the study (12 months), and exhibited no apparent reduction in muscle burdens over this period. Encapsulated larvae showed no obvious signs of degeneration in histological sections. Larval capsules were surrounded by infiltrates consisting of mature plasma cells and eosinophils. Macrophages were notably absent. Given the lack of a detectable serological response by 26 weeks p.i. and the persistence of infective muscle larvae for at least 1 year, parasite recovery methods are currently the only suitable detection assays for both meat inspection and epidemiological studies of Trichinella infection in the horse.     

Epidemiology of trichinellosis in Mexico, Central and South America

Trichinella species are widely distributed throughout the world and are found in a large number of carnivorous animals, humans and incidental hosts. The data presented in this review show that Trichinella infection has been reported in both humans and animals in Mexico, Argentina and Chile since the end of the 19th century, and more recently in Bolivia. This parasitic infection is still a public health problem in countries such as Argentina and Chile. Although efforts have focused on the control and prevention of trichinellosis in these countries, there were still human cases and outbreaks of trichinellosis reported. Diagnosis of infection in animals such as pigs still includes, in many endemic areas, the use of trichinoscopy. In Argentina, however, artificial digestion has been recently introduced in slaughterhouses to detect Trichinella infection in pigs. In some endemic areas in Mexico, the use of serological assays for human trichinellosis and pig infections have resulted in improved detection. Most of the outbreaks of human trichinellosis in Mexico, Argentina and Chile have occurred as a result of the consumption of undercooked pork or pork products from animals raised under poor hygienic conditions and which are clandestinely slaughtered. In several studies, rats, dogs and cats have been found to be infected with Trichinella and may be considered a risk for transmission of the infection to pigs or other animals intended for human consumption. Another potential source of transmission of Trichinella to humans is horsemeat; however, information related to horse trichinellosis in Latin-American countries is scarce. In most cases the etiological agent of human trichinellosis in Central and South America has been reported to be Trichinella spiralis; however, only in a few cases has the parasite species been properly identified. Based on the reports available, it is clear that there is a need to carry out better controlled epidemiological studies to determine the true prevalence of the infection in this region of the world. Also, more sensitive methods of diagnosis and improvements in conditions for pig production as well as better sanitary inspection systems, are needed for controlling and preventing transmission of trichinellosis in these countries.     

New patterns of Trichinella infection

Human and animal trichinellosis should be considered as both an emerging and reemerging disease. The reemergence of the domestic cycle has been due to an increased prevalence of Trichinella spiralis, which has been primarily related to a breakdown of government veterinary services and state farms (e.g., in countries of the former USSR, Bulgaria, Romania), economic problems and war (e.g., in countries of the former Yugoslavia), resulting in a sharp increase in the occurrence of this infection in swine herds in the 1990s, with a prevalence of up to 50% in villages in Byelorussia, Croatia, Latvia, Lithuania, Romania, Russia, Serbia, and the Ukraine, among other countries. The prevalence has also increased following an increase in the number of small farms (Argentina, China, Mexico, etc.) and due to the general belief that trichinellosis was a problem only until the 1960s. The sylvatic cycle has been studied in depth at both the epidemiological and biological level, showing the existence of different etiological agents (Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella nelsoni) in different regions and the existence of "new" transmission patterns. Furthermore, the role of game animals as a source of infection for humans has greatly increased both in developed and developing countries (Bulgaria, Canada, Lithuania, some EU countries, Russia, USA, etc.). The new emerging patterns are related to non-encapsulated species of Trichinella (Trichinella pseudospiralis, Trichinella papuae, Trichinella sp.), infecting a wide spectrum of hosts (humans, mammals including marsupials, birds and crocodiles) and to encapsulated species (T. spiralis, T. britovi, and T. murrelli) infecting herbivores (mainly horses). The existence of non-encapsulated species infecting mammals, birds and crocodiles had probably remained unknown because of the difficulties in detecting larvae in muscle tissues and for the lack of knowledge on the role of birds and crocodiles as a reservoir of Trichinella. On the other hand, it is not known whether horse and crocodile infections existed in the past, and their occurrence has been related to improper human behavior in breeding. The problem of horse-meat trichinellosis is restricted to France and Italy, the only two countries where horse-meat is eaten raw, whereas mutton and beef have been found to be infected with Trichinella sp. only in China.     

The sylvatic Trichinella cycle and its implications for Trichinella control in Germany

Trichinellosis is a food-borne, zoonotic disease caused by a parasitic organism. Pork containing muscle larvae represents the most important source of human trichinellosis. In Germany, each slaughtered domestic swine is systematically sampled and examined for Trichinella spp. European Union legislation (EC (No.) 2075/2005) condones the approach of a risk-oriented meat inspection for Trichinella in pigs which is accompanied by monitoring programmes for pig holdings and reservoir animals. Here we discuss the current epidemiological situation of Trichinella in the sylvatic cycle in Germany and the implications for the implementation of risk-based sampling.     

First isolation of Trichinella britovi from a wild boar (Sus scrofa) in Belgium

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.     

Influence of methods for Trichinella detection in pigs from endemic and non-endemic European region

A total of 1401 German and 226 Croatian pigs raised either indoors or outdoors were tested for Trichinella infection by direct and indirect detection methods. A 10 g sample of diaphragm were examined for muscle larvae by the artificial digestion method; the species was determined by multiplex polymerase chain reaction (PCR). For detection of anti-Trichinella IgG, serum samples diluted 1:100, and meat juice samples diluted 1:10, were tested by enzyme-linked immunosorbent assay. All German pigs and those Croatian pigs raised indoors proved to be Trichinella-negative by all methods. Muscle larvae were detected in a total of eleven of the Croatian pigs, which were raised on small outdoor farms. For eight isolates, PCR results demonstrated that recovered larvae were Trichinella spiralis. Anti-Trichinella-IgG was detected in serum and meat juice of digestion positive animals when the worm burdens exceeded 0.38 larvae per gram of muscle. Positive results in Croatian pigs indicate a higher risk of infection for outdoor farming in areas where Trichinella is endemic. Results of direct and indirect detection were compared and are discussed with special regard to specificity and sensitivity of methods.     

Proposals for designing the food chain information for the implementation of the risk-oriented ante- and post-mortem meat inspection

The recent crises in the meat industry due to meat-associated risks such as salmonella, nitrofen and dioxin prove that the traditional ante- and post mortem inspection of slaughter animals and carcasses is not any longer able to recognise and prevent the risks of today. Therefore, the EU Commission has issued Reg. (EC) 853/2004 and Reg. (EC) 854/2004 that regulate the transition of the traditional meat inspection, which demands inspecting each individual carcass in the same way, to a risk-based meat inspection, which is using relevant pieces of information about the previous production stages for making risk-based decisions on the intensity of the inspection of slaughter pig batches. The new EU legislation is not any longer prescribing exactly the inspection procedure, but defines the food safety goals. The consequence is that there are still various ideas and opinions on how to implement a reasonable risk-based meat inspection, and, in particular, on how to design the "relevant food chain information". The present paper is describing the legal framework and the objectives of the risk-based meat inspection, and proposals for designing the food chain information for the implementation of the risk-based ante- and post-mortem meat inspection are made and discussed.     

Evaluation of the risk of transmission of Trichinella in pork production systems in Argentina

Recently, there has been interest in programs that certify pork production practices that minimize the risk of exposure of pigs to Trichinella spiralis. Certification might be useful for reducing the risk of human trichinellosis from pork in Argentina, but more information is needed on pig production practices and sources of Trichinella infection in Argentinian pigs. In this study, 21 pig farms were assessed for Trichinella infection including some farms using total and partial confinement management, and others with pigs raised exclusively outdoors. A total of 3224 muscle samples were collected from pigs raised on these farms and tested to determine the presence of T. spiralis larvae by artificial digestion. Serum samples from the same 3224 pigs were tested for antibodies to T. spiralis by ELISA. For each farm, a questionnaire was completed summarizing information about management factors and this information was used to assess risk factors for exposure of T. spiralis. Based on the results, pigs raised outdoors were more likely to be infected than pigs raised in total or partial confinement (p ≤ 0.05). Pigs fed waste products containing meat were 12.5 times more likely to be infected than pigs not fed waste containing meat (p < 0.01). The role played by rats in transmission of Trichinella is unclear; however, on farms with evidence of wild animals and access of pigs to wildlife carcasses, the prevalence of Trichinella infection was significantly higher. All pigs raised under good hygienic and sanitary conditions were negative for Trichinella infection by both artificial digestion and ELISA.     

Detection of Trichinella infection in food animals

The first part of this review article deals with classical methods used for the detection of Trichinella larvae in muscle samples of those animal species which are recognized as traditional sources of trichinellosis for human beings, as well as those species which are important for epidemiological reasons. Special consideration is given to the main applications of these methods (routine slaughter inspection, and epidemiological studies in reservoir animals), and to the major factors that may influence detection methods (sampling site, sample size). Historical, current and future aspects concerning national and EU legislation for Trichinella inspection are also presented. The latter part of this review is directed at serodiagnostic methods for the detection of Trichinella-specific antibodies in different animal species. Classical methods of serodiagnosis such as the complement fixation test and immunofluorescence antibody test are reviewed and the characteristics and performance of the ELISA are discussed. Factors dependent upon the animal species being tested or on components of the ELISA test system are considered. This paper also reviews systematic development of the ELISA in relation to improvements in test specificity and sensitivity. Additionally, remarks are made on implementing this test for surveillance and control programs in domestic pigs and wildlife.     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

Trichinella in horses: a low frequency infection with high human risk

After the initial report in 1976 of a trichinellosis epidemic caused by the consumption of infected horsemeat, 12 other outbreaks have been described in Europe. Since the first serious human outbreak several experiments have confirmed the susceptibility of horses to Trichinella species and the rapid disappearance of specific antibodies in this host that prevents the use of serological methods for routine screening. A review of the distribution of parasite burdens in muscles of naturally or experimentally infected horses indicates that the tongue is the most likely sample to contain detectable numbers of Trichinella larvae in low level infections. Requirements for testing of horsemeat are specified in legislation of the European Union, and other recommendations are published elsewhere. The EEC directives have evolved into very specific requirements which specify the testing of at least 5g of tongue, masseter or diaphragm per horse using a pooled digestion assay. More recently, France has revised the requirement for sample size to 10g for horsemeat originating from countries with high prevalence of Trichinella. To address the continuing outbreaks of human trichinellosis due to infected horsemeat, the development and implementation of a quality assurance system for testing is being considered.     

The need for implementation of International Commission on Trichinellosis recommendations, quality assurance standards, and proficiency sample programs in meat inspection for trichinellosis in Serbia

Implementation of methods to control inspection for Trichinella in meat recommended by International Commission on Trichinellosis (ICT), particularly the introduction of the quality assurance standards and proficiency panels for certified analysts is extremely important in Serbia and other countries where Trichinellosis is endemic. In spite of existing regulations, including the inspection of 0.5 g samples of diaphragm by the compression method or by artificial digestion of 1g samples, in Serbia 280 people were diagnosed with clinical trichinellosis after consumption of inspected meat during the period 2001--2002. These outbreaks, which occurred in the municipalities of Kumane, Surcin and Bogatic, were a consequence of inadequate application of inspection methods and insufficient education of some veterinary inspectors. The problem of inadequate veterinary inspection in Serbia can be overcome by strict application of the ICT recommendations for the control of Trichinella with specific emphasis on implementing the quality assurance system (QAS) and proficiency sampling (PS/--PP/panel).