Trich-tracker - a practical tool to trace Trichinella spiralis transmission based on rapid, cost-effective sampling of genome-wide genetic variation.

Molecular epidemiology using traditional sequencing has been notoriously difficult in inbred parasites due to a lack of genetic variation available for discriminating among parasites. Next generation sequencing techniques offer a solution to this problem by increasing the number of loci that can be sequenced. Here, we introduce Trich-tracker, a tool that makes efficient use of diagnostic variation distributed throughout the genome of Trichinella spiralis to more rapidly, and conclusively, resolve connections and distinctions among focal outbreaks of T. spiralis. In particular, we rapidly characterised genetic variation among a sample of parasites from Polish farms and wildlife, sampling genomic variation using double digest restriction site-associated DNA sequencing (ddRADseq). Approximately 400,000 bases of sequence were generated from each sample and shown to be distributed across the genome with single nucleotide polymorphisms occurring at a frequency of approximately one base in 10,000. Both phylogenetic and Bayesian clustering analyses indicated that ddRADseq genotypes formed distinct clusters for specific outbreaks and were quite distinct from wild boar samples. Two of the investigated outbreaks were more similar to each other than to other outbreak samples, suggesting a link between these outbreaks. Hence, the Trich-tracker procedure identified informative genomic variation which afforded unprecedented epidemiological resolution. Trich-tracker is very flexible tool, quickly and inexpensively mining genomes of even highly inbred populations of T. spiralis to support outbreak investigations. The simplicity of the entire procedure, and time and cost effectiveness of Trich-tracker support its practical application in ongoing Trichinella outbreaks. The discriminating power of this tool is tunable and scalable, allowing application in a variety of epidemiological contexts, and is easily adapted to other parasite systems.

The Disease Ecology, Epidemiology, Clinical Manifestations, and Management of Trichinellosis Linked to Consumption of Wild Animal Meat.

Historically, human trichinellosis was caused by Trichinella spiralis and transmitted to humans by consumption of undercooked domestic pork. Today, most cases of trichinellosis are caused by other Trichinella species and transmitted by consumption of raw or undercooked wild game meats. Given the increasing global prevalence of wild animal meat-linked trichinellosis, the objectives of this review are: 1) to describe the life cycle and global distribution of Trichinella worms; 2) to describe the changing epidemiology of trichinellosis; 3) to describe the clinical phases of trichinellosis; 4) to recommend the latest diagnostic tests; and 5) to recommend treatment and prevention strategies. Internet search engines were queried with keywords as subject headings to meet the objectives of this review. Although trichinellosis surveillance systems and laws regulating commercial pork production have limited T spiralis-caused trichinellosis in Europe and the United States, trichinellosis due to consumption of raw and undercooked wild boar and feral hog meat continues to occur throughout Southeast Asia. Trichinellosis due to consumption of raw or undercooked meats of other infected game, such as bear, deer, moose, and walrus, continues to occur worldwide. Only adherence to hygienic practices when preparing wild game meats and cooking wild game meats to recommended internal temperatures can prevent transmission of trichinellosis to humans. Wilderness medicine clinicians should be prepared to advise hunters and the public on the risks of game meat-linked trichinellosis and on how to diagnose and treat trichinellosis to prevent fatal complications.

Primary characterization and assessment of a T. spiralis antigen for the detection of Trichinella infection in pigs.

A clone, designated L20h-Ts3, was selected by immunoscreening of cDNA libraries of Trichinella spiralis worms collected 14h, 20h and 48h post-infection (p.i.) from mice intestines. L20h-Ts3 encodes the full-length of a conserved hypothetical protein of 13.1kDa involving putative interaction with the immune system. PCR analysis showed that L20h-Ts3 mRNA is constitutively expressed throughout T. spiralis life cycle and not restricted to intestinal stages. The L20h-Ts3 fusion protein was obtained in an Escherichia coli expression system and purified by Ni-affinity chromatography before inoculation into mice in order to produce polyclonal antibodies. Then, immunohistochemical study and Western blot analysis revealed its presence within the stichosome of T. spiralis and in excretory/secretory products strengthening a putative fundamental role for the parasite's survival such as host tissue invasion or modification of the host muscular cell phenotype. L20h-Ts3 fusion protein was recognized in Western blot as soon as 15-20 days p.i. by sera from pigs experimentally infected with 20,000 muscle larvae (ML) of T. spiralis. Thus, an indirect L20h-Ts3 ELISA was designed and evaluated using sera from experimentally infected pigs by comparison with the only ELISA currently available for trichinellosis purposes. A gain of precocity from 7 up to 14 days and detection up to 25 weeks p.i. was possible with the L20h-Ts3 ELISA offering a large window for trichinellosis detection. The L20h-Ts3 ELISA was less effective in the case of low infections in pigs. Nevertheless, these results show that the L20h-Ts3 ELISA has a real interest due to its precocity and stability of detection in time. The association of the L20h-Ts3 fusion protein with other antigenic proteins identified previously could appreciably improve the serological test and facilitate its standardization.

Assessment of selected biochemical parameters and humoral immune response of Nile crocodiles (Crocodylus niloticus) experimentally infected with Trichinella zimbabwensis.

Fifteen crocodiles were randomly divided into three groups of five animals. They represented high-infection, medium-infection and low-infection groups of 642 larvae/kg, 414 larvae/kg and 134 larvae/kg bodyweight, respectively. The parameters assessed were blood glucose, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT). The humoral immune response to Trichinella zimbabwensis infection was evaluated in all three groups by an indirect ELISA method. The results showed deviations from normal parameters of blood glucose, CPK, LDH, AST and ALT when compared with reported levels in uninfected reptiles. Contrary to studies involving mammals, hypoglycaemia was not observed in the infected groups in this study. Peak values of blood glucose were reached on post-infection (PI) Day 49, Day 42 and Day 35 in the high-infection, medium-infection and low-infection groups, respectively. Peak values of LDH and AST were observed on PI Day 56, Day 49 and Day 42 in the high-infection, medium-infection and low-infection groups, respectively. Peak values of CPK were observed on Day 35 PI in all three groups. Peak ALT values were reached on Day 56 in the high-infection group and on Day 28 PI in both the medium-infection and low-infection groups. No correlations between the biochemical parameters and infection intensity were observed. Peak antibody titres were reached on Day 49 PI in the medium-infection group, and on Day 42 PI in both the high-infection and low-infection groups. Infection intensity could not be correlated with the magnitude of the humoral immune response or time to sero-conversion. Results from this study were in agreement with results reported in mammals infected with other Trichinella species and showed that antibody titres could not be detected indefinitely.

The economic impact of pig-associated parasitic zoonosis in Northern Lao PDR.

The parasitic zoonoses human cysticercosis (Taenia solium), taeniasis (other Taenia species) and trichinellosis (Trichinella species) are endemic in the Lao People's Democratic Republic (Lao PDR). This study was designed to quantify the economic burden pig-associated zoonotic disease pose in Lao PDR. In particular, the analysis included estimation of the losses in the pork industry as well as losses due to human illness and lost productivity. A Markov-probability based decision-tree model was chosen to form the basis of the calculations to estimate the economic and public health impacts of taeniasis, trichinellosis and cysticercosis. Two different decision trees were run simultaneously on the model's human cohort. A third decision tree simulated the potential impacts on pig production. The human capital method was used to estimate productivity loss. The results found varied significantly depending on the rate of hospitalisation due to neurocysticerosis. This study is the first systematic estimate of the economic impact of pig-associated zoonotic diseases in Lao PDR that demonstrates the significance of the diseases in that country.

Quantitative risk assessment for the annual risk of exposure to Trichinella spiralis in imported chilled pork meat from New Zealand to Singapore.

AIMS: To determine the annual likelihood of exposure to an infectious dose of Trichinella spiralis from consuming imported pork meat from New Zealand to Singapore. METHODS: Input values specific for chilled pork meat imported into Singapore from New Zealand were used in a quantitative risk-assessment model. The model, designed to allow any combination of importing and exporting countries, was divided into two components, viz the release assessment, and the exposure assessment that assessed the annual risk of exposure to the consumer (ARC). The former estimated the likelihood that a contaminated fresh meat product from New Zealand would arrive at Singapore's border, and took into consideration the prevalence of disease on different types of farms. The latter determined the likelihood over a year that a person in Singapore would consume one or more servings of imported fresh meat from New Zealand that contained a burden of greater than or equal to one larva(e) of T. spiralis per gram after preparation for consumption. RESULTS: The ARC for offal was 2.41 x 10(-7), which was below the pre-selected safety threshold of 1.00 x 10(-6). The ARC for lean meat was 2.39 x 10(-5), which was above the acceptable safety threshold. CONCLUSIONS: The study demonstrated that continued routine testing at slaughter is unnecessary for pig offal produced commercially, and provided a model with which to further assess management of the risk of exposure to T. spiralis in lean meat. CLINICAL RELEVANCE: The potential of Trichinella species to cause disease in humans is a public health concern, and has created adverse effects on the international trade of fresh lean meat without regard to the surveillance measures employed by particular pork-producing countries.

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.

Re-emergence of trichinellosis in southeastern Europe due to political and economic changes.

The countries of southeastern Europe including the Balkan region and bordering countries - Albania, Bulgaria, Bosnia and Herzegovina, Croatia, Greece, Hungary, Macedonia, Romania, Serbia and Montenegro, Slovenia, and the European part of Turkey - occupy a very important strategic position and represent a land bridge between Europe and Asia. In the majority of southeastern European countries, cases of trichinellosis among the human and animal populations were described in the late 19th or early 20th centuries. Trichinella infections among wildlife were also described in the aforementioned countries. Today, the prevalence of trichinellosis is different between the Balkans and bordering countries. A high prevalence of trichinellosis in domestic animals and humans has been reported in Bulgaria, Serbia and Montenegro, Romania and Croatia. A moderate prevalence was found in Bosnia and Herzegovina. In Hungary, human trichinellosis has not been present for a long period of time. However, sporadic cases were recorded in swine over the last 2 years. Trichinellosis has not been found among domestic animals and humans in Greece and Macedonia in recent years while in Turkey and Slovenia human trichinellosis is sporadic. The re-emergence of trichinellosis is connected with the changes in the social and political systems in Bulgaria and Romania. In Serbia and Montenegro as well in Croatia, however, a re-emergence of trichinellosis was due not only to political and social changes but also to wars that took place in these countries during the last years of the 20th century. Social, economic and political factors responsible for the re-emergence of trichinellosis in southeast European countries are discussed in this communication.

Application and assessment of a dipstick assay in the diagnosis of hydatidosis and trichinosis.

The aim of the present work was to apply and evaluate a dipstick assay for the serodiagnosis of human hydatidosis as well as human and experimental trichinosis using camel hydatid cyst fluid (HCF) and Trichinella spiralis muscle larval (TSML) antigens, respectively, and compare this to enzyme-linked immunoelectrotransfer blot (EITB) and Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA). Sera samples were collected from patients with confirmed hydatidosis and trichinosis and with other parasitic diseases as well as from normal healthy individuals. Also, sera samples were collected from mice experimentally infected with T. spiralis which were sacrificed at different time points post-infection (PI). HCF and TSML antigens were used in EITB after separation and characterization of their antigenic components using 5-22.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing condition. For the diagnosis of hydatidosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100, 91.4 and 95.1% while those of FAST-ELISA were 96.2, 100 and 98.4%, respectively. For the diagnosis of human trichinosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100% while those of FAST-ELISA were 85.7%. FAST-ELISA proved to be more sensitive in the early diagnosis of experimental T. spiralis infection (100% sensitivity from the second week PI) than the dipstick and EITB (100% sensitivity from the third week PI). All tests retained their sensitivity till the 12th week PI. Since the dipstick assay is extremely easy to perform with a visually interpreted result within 15 min, in addition to being both sensitive and specific, the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment and the technological expertise needed for EITB and FAST-ELISA.

Assessment of host resistance to Trichinella spiralis in mice following preinfection exposure to 2,3,7,8-TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been reported to decrease host resistance to a variety of infectious agents when exposure occurs prior to infection. Resistance to viral infection has been observed at doses as low as 0.1 microgram TCDD/kg body wt, well below the thymolytic dose in mice. In the present study, female B6C3F1 mice were exposed to a single intraperitoneal injection of 0, 0.1, 1.0, 10.0, or 30.0 micrograms TCDD/kg 7 days prior to infection to determine the effects of TCDD exposure on resistance to the nematode parasite Trichinella spiralis. Exposure to 10 or 30 micrograms TCDD/kg delayed adult parasite elimination from the small intestine. Significantly more larvae were released by female parasites and greater numbers of encysted larvae were recovered from the muscle of mice exposed to TCDD. Proliferative responses of splenocytes and mesenteric lymph node cells stimulated with T. spiralis antigen were significantly suppressed at exposure levels of TCDD > or = 1.0 microgram/kg 7 days after infection and in splenocytes only at 14 days after infection, demonstrating the greater sensitivity of proliferative responses to TCDD exposure than actual host resistance to Ts infection. Suppressed proliferation was observed at doses which produced TCDD concentrations > or = 0.2 pmol/g of lymphoid tissue on Day 7 of infection. In addition, it was determined that infected mice had higher TCDD levels than noninfected mice given the same dose. These results suggest an interaction between TCDD exposure and infection, i.e., that exposure to TCDD altered the host response to infection, while infection delayed elimination of TCDD from the host.