Seroprevalence of Toxoplasma gondii and direct genotyping using minisequencing in free-range pigs in Burkina Faso.

BACKGROUND: Swine are a major source of meat for humans. As such, they can play an important role in the epidemiology of human toxoplasmosis. Therefore, we performed an epidemiological study to determine the prevalence and genotypes of Toxoplasma gondii in Burkina Fasan swine. METHODS: The prevalence of T. gondii infection was evaluated in a 3-month prospective study at the slaughterhouse of Bobo-Dioulasso, Burkina Faso. Anti-Toxoplasma IgG titers were determined on meat juices from pig diaphragms using a commercially available ELISA assay. The DNA was extracted from 25mg of heart biopsies of seropositive animals (IgG ≥50% of the control) and the presence of T. gondii DNA was detected using a quantitative PCR assay. Genotyping was performed directly on DNA from PCR-positive biopsies using high-resolution melting and minisequencing analyses of the repeated B1 gene. RESULTS: The prevalence of carcasses positive for anti-Toxoplasma IgG was 29% (87/300) with no difference according to sex and age in contrast to the village of origin (p=0.018). Of the 87 seropositive animals, two were PCR positive (parasitic load at 64 and 128 parasites/mg of heart biopsy). Two new genotypes belonging to Type II and Type III and different from the genotypes previously described using minisequencing were identified. CONCLUSION: Our study provides the first T. gondii seroprevalence data in Burkina Fasan swine. In addition, this direct typing method suggests diversity of the T. gondii genotypes circulating in domestic animals in Burkina Faso. This needs to be confirmed on a wider sampling of subjects.

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.

Freeze-tolerance of Trichinella muscle larvae in experimentally infected wild boars.

Freeze-tolerance of encapsulated Trichinella muscle larvae (ML) is mainly determined by Trichinella species, but is also influenced by host species, the age of the infection and the storage time and temperature of the infected meat. Moreover, the freeze-tolerance of the encapsulated species appears to be correlated to the development of thick capsule walls which increases with age. An extended infection period and the muscle composition in some hosts (e.g. herbivores) may provide freeze-avoiding matrices due to high carbohydrate contents. The present experiment compares freeze-tolerance of Trichinella spiralis and Trichinella britovi ML in wild boar meat 24 weeks post inoculation (wpi). Three groups of four wild boars were infected with 200, 2000 or 20,000 ML of T. britovi (ISS 1575), respectively. Additionally, three wild boars were inoculated with 20,000 ML of T. spiralis (ISS 004) and two animals served as negative controls. All wild boars were sacrificed 24 wpi. Muscle samples of 70 g were stored at -21°C for 19, 30 and 56 h, and for 1-8 weeks. Larvae were recovered by artificial digestion. Their mobilities were recorded using Saisam(®) image analysis software and their infectivities were evaluated using mouse bioassays. Samples frozen for 19, 30 and 56 h allowed recovery of mobile ML, but samples frozen for 1 or 2 weeks did not. Correspondingly, only T. spiralis and T. britovi larvae isolated from wild boar meat frozen for 19, 30 and 56 h established in mice. This study showed that freezing at -21°C for 1 week inactivated T. spiralis and T. britovi ML encapsulated in wild boar meat for 24 weeks.

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.

Use of proficiency samples to assess diagnostic laboratories in France performing a Trichinella digestion assay.

Routine diagnosis of animal trichinellosis for food safety and trade relies on a method of artificial digestion to free Trichinella muscle larvae from meat for subsequent identification by microscopy. As part of a quality control system, the French National Reference Laboratory (NRL) initiated ring trials to determine the sensitivity of the test performed in the 72 routine diagnostic laboratories in France. A method was devised to obtain calibrated meat samples containing known numbers of capsules with Trichinella spiralis muscle larvae. This method was based on an incomplete artificial digestion of Trichinella-infected mice carcasses to allow the collection of intact Trichinella capsules. Capsules were placed into a meatball of 100 +/- 2 g of pork and horsemeat to produce proficiency samples. Three categories of samples were prepared: small (3 to 5 capsules), medium (7 to 10), and large (12 to 15). The sensitivity was expressed as the percentage of muscle larvae recovered from each proficiency sample. Reproducibility was tested with ring trials organized between two NRLs (France and Canada), and a reference sensitivity of 84.9% was established. National ring trials were then organized in France, with the 72 routine diagnostic laboratories each receiving four proficiency samples per session. After five sessions, an improvement in the digest test sensitivity was observed. Results at the fifth session indicated sensitivities of 78.60% +/- 23.70%, 81.19% +/- 19.59%, and 80.52% +/- 14.71% muscle larvae for small, medium, and large samples, respectively. This study supports the use of proficiency samples to accurately evaluate the performance of routine diagnostic laboratories that conduct digestion tests for animal trichinellosis diagnosis.