Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK.

Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNA was detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR-RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6·0 to 44·4% depending on the host. PCR-RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Detection of Neospora caninum in wild carnivorans in Great Britain.

Samples of brain and other tissues were collected from 99 ferrets (Mustela furo), 83 red foxes (Vulpes vulpes), 70 European polecats (Mustela putorius), 65 American mink (Neovison vison), 64 Eurasian badgers (Meles meles) and 9 stoats (Mustela erminea), from around Great Britain. DNA was extracted from approximately 1g of tissue and tested by specific nested ITS1 PCR for Neospora caninum. The results from the PCR demonstrated that Neospora specific DNA was detected in all species of wild carnivorans with the exception of the stoats (0/9). Neospora DNA positive samples were detected in: polecats 18.6% (13/70), badgers 10.9% (7/64), ferrets 10.1% (10/99), foxes 4.8% (4/83) and mink 4.6% (3/65). In the badgers N. caninum DNA positive samples were found in brain (n=2), liver (n=2) and neck muscle (n=3). Selected positive ITS1 DNA sequences were submitted to Genbank. Sequence UKwildlife1 (accession number JX857862) was found in two badgers, whilst UKwildlife2 and UKwildlife3 (accession numbers JX857863 and JX857864 respectively) were found in ferrets, all three sequences demonstrated point mutations at a single base, while sequence UKwildlife4 (accession number JX857865) was found in all the species that tested positive and showed complete identity when compared against published reference sequences for: N. caninum (Nc Liverpool isolate, EU564166). Our data shows that almost all the wild carnivoran mammal species tested are intermediate hosts for N. caninum and are therefore capable of acting as reservoirs of infection for other species. These species could also act as useful sentinel species, demonstrating the presence of the parasite in particular geographical and environmental locations.

A diagnostic study of Echinococcus multilocularis in red foxes (Vulpes vulpes) from Great Britain.

Alveolar echinococcosis is caused by a parasitic tapeworm Echinococcus multilocularis and is a serious disease with high fatality in humans. The definitive primary host is the red fox (Vulpes vulpes) but domestic animals (dogs and to a lesser extent cats) as well as several genera of rodents can also be infected with the parasite. There is, to date, no evidence of indigenous cases of E. multilocularis in Great Britain (GB) but in most of continental Europe the parasite is considered to be endemic and/or slowly spreading. All pet dogs entering the United Kingdom (UK) under the pet travel scheme (PETS) are therefore currently treated with an anthelmintic effective against Echinococcus spp. Surveillance of red foxes is required to demonstrate disease freedom and maintain this regulation to prevent further geographical spread of the parasite to free areas within the EU. A study of 588 wild red foxes collected from across Great Britain (GB) between October 1999 and November 2000 found no Echinococcus spp. This report describes a further study of GB foxes collected predominately during 2005 and 2006. Fox faecal samples (n=384) were examined for both E. multilocularis and Echinococcus granulosus using an egg isolation procedure followed by PCR method, based on published primer sets. A non-specific primer set that amplifies Taenia spp. as well as Mesocestoides, Dipylidium and Diphyllobothrium was also included in the assay to validate the test procedure as these parasites are expected to be more common in wild fox populations. All faecal samples tested negative for both E. multilocularis and E. granulosus but results for approximately 35% of the samples indicated the presence of Taenia spp. or other closely related cestodes. This data contributes to the evidence that suggests that E. multilocularis is not present in mainland Britain and justifies the requirement for ongoing surveillance to demonstrate disease freedom.

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.

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.

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.