Screening for infection of Trichinella in red fox (Vulpes vulpes) in Denmark

A total of 6141 foxes (Vulpes vulpes) were examined for infection with Trichinella. The foxes were killed in Denmark during the hunting season 1995-1996 and 1997-1998; 3133 and 3008, respectively. Foxes included in the investigation came from throughout the country with the exception of the island of Bornholm. The right foreleg from each fox was submitted for investigation. The legs were stored at -20 degrees C for 3-10 months prior to examination. Following thawing, muscle tissue (10 g) from each leg was examined by trichinoscopy and by a pepsin-HCl digestion technique. In 1995-1996, three foxes were found positive corresponding to a prevalence of 0.001. Each of the infected foxes harboured an extremely low infection, i.e. about one larva per 10 g muscle tissue. It was not possible to obtain sufficient larval material for species identification. All three foxes were shot in the vicinity of a small village in the north-western part of Denmark. In 1997-1998 no Trichinella cases were found. The results, compared with previous studies, indicate that the prevalence of infection of Trichinella sp. among wild living foxes in Denmark is very low. This is further supported by the fact, that no infection of Trichinella sp. has been found in slaughtered pigs in Denmark for more than 65 years, which suggests that the infection pressure is very low. Considering the facts above we conclude that the risk of Trichinella infections is negligible in intensive indoor pig production units in Denmark whereas high local prevalence of Trichinella infections in the wildlife might constitute a serious risk for the expanding outdoor pig production.     

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.     

Towards a standardised surveillance for Trichinella in the European Union

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.     

Comparison of two antigens for demonstration of Trichinella spp. antibodies in blood and muscle fluid of foxes, pigs and wild boars

For the surveillance of trichinellosis, the digestion method is reliable but also labour intensive. The serological methods for the detection of Trichinella-specific antibodies using ELISA offer a sensitive and relatively specific alternative. For serological studies, sera or plasma from blood samples are the most common source of antibodies, but although the concentration of antibodies is approximately 10-fold lower, muscle fluid can be a good alternative particularly for testing of wildlife samples. In the present study, an indirect ELISA technique was evaluated on both sera and muscle fluids from experimentally infected foxes, pigs, and wild boars using both excretory/secretory (E/S) antigens and a synthetic glycan antigen, beta-tyvelose. Although the synthetic antigen appears to be less sensitive than the E/S antigens, Trichinella-specific IgG antibodies were detected in both serum samples and muscle fluid samples from pigs, wild boars and foxes infected at levels which would be important for food safety or represent a significant reservoir for further transmission.     

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.     

Report of Trichinella spiralis in a red fox (Vulpes vulpes) in Northern Ireland

No systematic studies of the occurrence of Trichinella in wildlife have been carried out in Northern Ireland (NI) in recent years, and the last reports of trichinellosis in livestock and human outbreaks in NI date back to 1979 and 1945, respectively. In this study, covering the period 2003/2004 and 2007/2008, a total of 443 red foxes (Vulpes vulpes) were collected throughout the country and screened for trichinellosis using a modified muscle digest method. One examined animal was found to be infected with larvae from Trichinella spiralis, indicating a national prevalence in NI of Trichinella in foxes of 0.2%. This prevalence compares well to the findings reported from the bordering Republic of Ireland [Rafter, P., Marucci, G., Brangan, P., Pozio, E., 2005. Rediscovery of Trichinella spiralis in red foxes (Vulpes vulpes) in Ireland after 30 years of oblivion. J. Infect. 50, 61–65] and could be a further indication for a sylvatic Trichinella life cycle existing independently from the domestic cycle.     

Prevalence of zoonotic important parasites in the red fox (Vulpes vulpes) in Great Britain

A national necropsy survey of red foxes was carried out across Great Britain to record Echinococcus, Trichinella and Toxoplasma. The survey did not record directly, or indirectly using coproantigen/PCR tests, evidence for the presence of Echinococcus multilocularis in 588 animals, although E. granulosus was suspected in six animals. Parasitological evidence for Trichinella spp. could not be found in 587 fox muscle digests, and a specific PCR test also failed to detect Toxoplasma in a sub-set of 61 random fox tongue biopsies. The upper 95% confidence interval for the above parasites was 0.60% (E. multilocularis), 0.60% (Trichinella spp.) and 5.6% (Toxoplasma). The commonest gut parasites were the hookworm Uncinaria stenocephala (41.3%) and the ascarid Toxocara canis (61.6%). This study also reports the second occurrence of Trichuris vulpis in Great Britain.     

The epidemiology of animal trichinellosis in China

The epidemiology of animal trichinellosis in China based mainly upon original Chinese literature published between 1937 and 2004 is reviewed. The seroprevalence of Trichinella infection in herbivores was 0.7% (2/300) in cattle and 0.8% (4/500) in sheep. The prevalence of trichinellosis in naturally infected cattle was 1.2% (2/163). Trichinella larvae were detected in 1.4% (3/215) of sheep and in 2.1% (1/47) of beef cattle sold at markets. Canine trichinellosis was recorded in 13 Provinces, Autonomous Regions or Municipalities (P/A/M) and the average prevalence of the infection in dogs slaughtered in abattoirs was 16.2% (5654/34,983) ranging from 1.2% to 44.8%, with the highest prevalence located in northeast China. The prevalence in dog meat sold at markets was 3.5% (988/27,898) in 5 P/A. Feline Trichinella infection was reported in 10 P/A/M. The prevalence of Trichinella infection in rats varied from 1.1% (51/459) to 15.1% (50/332). Trichinella larvae were detected in 1.5% (9/587) of house rats (Rattus norvegicus) as well as in 0.8% (3/369) of wild rats (Apodemus chevrieri), and the infection was recorded also in other wildlife (foxes, bears, wild boar, weasels, raccoon dogs, muntjak and bamboo rats). Trichinella larvae were detected in 2.6% (4/156) of weasels (Mustela sibirica), 1.5% (2/135) of shrews (Tupaia belangeri) and 7.7% (1/13) of moles (Parascapter leucurus). All Trichinella isolates from domestic pigs were identified as T. spiralis. Some Trichinella isolates from dogs in north-eastern China were identified as T. nativa, which has muscle larvae that are highly resistant to freezing. Twenty-seven outbreaks of human trichinellosis associated with mutton, dog and game meat occurred in China between 1964 and 2004, but the quarantine of Trichinella larvae in such meat is not mandatory in China at present.     

Autochthonous and imported Trichinella isolates in Germany

The study of Trichinella isolates from wildlife in Germany revealed the presence of Trichinella spiralis and Trichinella britovi in wild boars and foxes. T spiralis was detected in meat products imported from Spain, which is one of the two endemic areas of domestic trichinellosis in the European Union: It was also detected in meat from a grizzly bear marketed in Alaska, and Trichinella nativa was detected in a polar bear from the Berlin Zoo. These results stress the importance of examining for Trichinella live animals and meat products imported to Germany from both EU and non-EU countries. Furthermore, carnivores from Arctic regions that are born in the wild and placed in zoos can represent a risk for the introduction of the freeze-resistant species of Trichinella in a new region if, once the animal dies, the carcass is not properly destroyed.     

Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology

The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.     

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.     

Survey on porcine trichinellosis in Ecuador

A survey on porcine trichinellosis was organised in Ecuador between 2000 and 2003. Blood samples were taken in slaughterhouses (study 1, n=2000; study 2, n=331) and in a remote village where pigs are free roaming (study 3, n=646) and examined by ELISA using excretory/secretory (E/S) antigens. Seven samples (0.35%) in study 1 and none of the samples of study 2 were serologically positive. Thirty-seven (5.72%) village pigs tested positive by E/S ELISA in study 3. Sero-positive results by the E/S ELISA in study 1 were confirmed by ELISA using beta-tyvelose antigen, and by immunoblot. Muscle samples taken from pigs slaughtered in the abattoir (study 2) and from animals that showed a positive serology in study 3 were examined by trichinoscopy and artificial digestion. These techniques failed to demonstrate the presence of muscle larvae. The results of this survey need confirmation, but suggest that Trichinella is present in Ecuador; however, prevalence and parasite burdens are likely to be very low. The likelihood of detecting trichinellosis are higher in traditional settings than in pigs raised on improved farms.     

Trichinellosis in Argentina: an historical review

In Argentina, Trichinella infection in pigs is endemic. The first report of human trichinellosis in Argentina was from 1898 in Buenos Aires. The number of human cases increased from 908, between 1971 and 1981, to 6,919, between 1990 and 2002. In pigs slaughtered in official establishments, the prevalence of Trichinella infection was 0.46% in 1914 and 0.01--0.03% during the period 1990--2004. T. spiralis is typically found in the domestic cycle that includes pigs, humans and rodents. Trichinella spp. from a sylvatic cycle has also caused human outbreaks resulting from the consumption of meat from puma, armadillo and wild boar. European migration to Argentina (principally Spanish and Italian) during the first years of the 20th century brought the tradition of preparing and eating raw sausages. This increased the risk of human exposure to Trichinella. Detection in pigs was initially made at slaughter by compression of muscle tissue (trichinoscopy) and continued this way until 1996, when artificial digestion was adopted for use in preventing human trichinellosis in Argentina. The following report synopsizes the evolution of trichinellosis in Argentina over the past century.     

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.     

Comparison of trichinelloscopy with a digestion method for the detection of Trichinella larvae in muscle tissue from naturally infected pigs with low level infections

The identification of Trichinella infection in pigs in Croatia has traditionally been done by inspection of individual carcasses. In response to outbreaks of human trichinellosis in the last decade, the Croatian Ministry of Agriculture and Forestry instituted compulsory trichinelloscopic examination of tissue from both commercially and privately slaughtered swine. The purpose of this study was to compare trichinelloscopy and artificial digestion for use in samples containing low numbers of larvae. Each assay was used to test 1,769 field positive samples, 290 of which contained 6 or less larvae per gram of muscle tissue. The sensitivity and specificity of trichinelloscopy with 6 or less l pg was 43.4 and 88%, respectively. kappa-Value as a measure of agreement between trichinelloscopy and artificial digestion was 0.27%. It is noteworthy that a considerable number of the 103 (52%) negative animals on trichinelloscopy contained>or=6l pg which is enough to cause clinical trichinellosis. These findings support other studies that indicate trichinelloscopy is not a method of choice and that it is necessary to implement more sensitive procedures such as artificial digestion.     

Development and evaluation of an immunochromatographic strip for trichinellosis detection

In the present study, an immunochromatographic strip was developed for the serological detection of trichinellosis in swine. In the strip, the excretory-secretory (ES) antigens of Trichinella labelled with colloidal gold was used as the detector, and the staphylococcal protein A (SPA) and goat anti-ES antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. The evaluation of the strip was performed by comparing 60 clinical positive blood samples detected by the artificial digestion method with 46 serum samples from pigs infected with parasites other than Trichinella and 30 serum samples of parasite-free healthy pigs. The strip was shown to be of high specificity and sensitivity that were closely correlated with those of ELISA. Furthermore, the dipstick assay based on the strip is rapid (10 min) and easy to perform with no requirement of special skill, reagent or equipment. This suggests the immunochromatographic strip is an acceptable alternative to be used in clinical laboratories lacking specialized equipment as well as for field diagnosis.     

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.     

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.     

Serological evidence of Trichinellosis in local pigs of Nepal

In Nepal, animal husbandry is a major source of income. Pig husbandry is practiced in rural, peri-urban, and urban communities. Free ranging "back yard" pigs and the practice of feeding offal is a very common management practice which potentially allows for the transmission of trichinellosis; however, this zoonosis has never been reported from this region. A total of 425 serum samples were collected from local pigs. These were initially screened by ELISA after which positive samples were examined by Western blot. This procedure identified two samples which had clear specific bands for Trichinella; however, muscle samples tested by HCL-pepsin digestion were found to be negative. If these highly specific serological analyses are confirmed, this would be the first report of trichinellosis in Nepal and a prevention program should be initiated to limit the access of pigs to open garbage dumps which exist both in towns and on farms.     

Destruction of Trichinella spiralis spiralis during the preparation of the "dry cured" pork products proscuitto, proscuittini and Genoa salami

Genoa salami, proscuittini and proscuitto were prepared from pork carcasses that were heavily infected experimentally with Trichinella spiralis spiralis. Genoa salami was prepared with salt concentrations of 2.0%, 2.75% and 3.3%. Proscuitto was prepared by two procedures approved by Agriculture Canada. At various times postpreparation, samples of the various cured products were taken and examined by pepsin digestion and rat bioassay for the presence of viable trichinae. Water activity and pH of the cured meat were also determined. Curing of the various products was shown to destroy the Trichinella larvae. Pepsin digestion revealed that larvae progressively became loosely coiled, uncoiled and more subject to digestion (ghost larvae) during the curing process. Rat bioassay revealed the presence of viable trichinae in the proscuitto prepared using a sodium chloride salt mixture at day 34 but not at day 48 postpreparation. All other bioassays carried out on Genoa salami between 13 and 42 days postpreparation, on proscuittini between days 27 and 69 and on proscuitto between days 34 and 69 were negative for viable trichinae. Under the conditions of this study, preparing Genoa salami with salt concentrations as low as 2% did not appear to affect the destruction of Trichinella larvae.