Catherine Perret

An innovative survey underlining the significant level of contamination by Toxoplasma gondii of ovine meat consumed in France

Consumption of sheep meat presents a risk of human contamination by Toxoplasma gondii. A nationwide study was conducted in France to evaluate the prevalence of Toxoplasma in fresh ovine meat. A sampling procedure was established to guarantee the representativity of consumption. As is the case for meat consumed, half of the samples were from France and half were imported from other countries. Animals were selected according to their age, as lamb (<12months) represents 90% of the meat consumed. Available data for French samples allowed the selection of 16 districts distributed in seven areas according to their density of production. Diaphragms and hearts from 433 sheep were collected. Diaphragms were collected from 398 imported carcasses. Fluids from hearts and diaphragms were tested serologically. All hearts were bioassayed in mice and parasite isolates were genotyped using PCR-restriction fragment length polymorphism and microsatellite markers. Prevalence estimates were calculated, taking into account uneven distribution of production and age. For French meat, the effect of area, age and their interactions was evaluated. The overall estimate of Toxoplasma seroprevalence was 17.7% (11.6-31.5%) for lambs and 89% (73.5-100%) for adults (P<0.0001). No significant difference was observed between imported and French meat. In France, seroprevalence in lambs showed an increasing North-western to Southern gradient. The proportion of French carcasses carrying live parasites according to bioassay results was estimated at 5.4% (3-7.5%) (45 genotype II; one genotype III). This study offers an accurate drawing of the toxoplasmosis pattern amongst sheep consumed in France and a model for a zoonosis hazard control survey.

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.

Development and Evaluation of a Western Blot Kit for Diagnosis of Human Trichinellosis

ABSTRACT We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.

Cloning and analysis of a Trichinella britovi gene encoding a cytoplasmic heat shock protein of 72 kDa.

A gene encoding a protein of 646 amino acid residues with a molecular mass of 71.3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb). The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum. Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage). Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm. The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella.

Characterization of eleven antigenic groups in Trichinella genus and identification of stage and species markers.

Several monoclonal antibodies (Mabs) were raised against the L1 muscle stage (L1M) of Trichinella spiralis (Ts) and Trichinella pseudospiralis (Tp). Western blot analysis of various antigenic preparations established that Mabs described by different authors recognized 8 antigenic fractions (TSL1-TSL8) in crude extracts of infective larvae. The TSL1 fraction was immunodominant and present on the cuticle of different parasite stages. Mabs against Trichinella T5 (T5) and Ts were selected in order to extend the previous studies to another Trichinella phenotype. Only 35% of the selected Mabs recognized linear epitopes and 71% reacted with soluble or excretory-secretory antigens in a dot blot procedure and ELISA test. The targets of the Mabs were identified by immunoprecipitation with [35S]methionine-labelled L1M worm lysate. Mabs prepared from mice immunized with the whole parasite (T5) recognized a wider panel of antigens in different parasitic organs. Seven antigenic structures were distinguished on the cuticle and several epitopes were identified in the gut, haemolymph and stichocytes. Eleven antigenic groups were established according to their indirect immunofluorescence pattern on cross-sections of the worm. Monoclonal antibodies raised against Ts soluble antigen mainly recognized epitopes in stichocytes and on the cuticle surface. All the selected Mabs recognized T5 and Trichinella britovi (Tb) strengthening the link between these 2 species. Four Mabs were used to differentiate antigenic structures among 6 Trichinella phenotypes and to develop a new tool to follow gene flow within the Trichinella genus.

New strategy for the survey of Toxoplasma gondii in meat for human consumption.

Monitoring of Toxoplasma infection in animals destined for human consumption is a great challenge for human toxoplasmosis prevention. This study aimed to compare results obtained from a naturally infected population of sheep using different tests and targeting an original matrix: meat samples and muscle fluids collected at the slaughterhouse. A commercial ELISA test was performed on diaphragm fluids from 419 ovine carcasses collected at the slaughterhouse. A MAT (modified agglutination test) was performed on heart fluids obtained from the same animals. In addition, all hearts were bioassayed in mice. Serological test agreement, the relative sensitivity of ELISA MAT and mouse bioassay as well as a correlation between titres and parasite isolation probability were statistically evaluated. The overall agreement (kappa coefficient=0.64) of ELISA on diaphragm fluids and MAT on heart fluids is substantial and subsequently both tests can be used for epidemiological studies. Relative sensitivity was higher for MAT performed on cardiac fluids (90%) than ELISA on diaphragm fluid (61%). For both serological tests, relative sensitivity is lower in lambs younger than 12 months. Relative sensitivity of mouse inoculation was 42%. A significant correlation was obtained between increasing MAT titres and probability to isolate live parasite from the heart. When the fluid titre was higher than 1:16, parasites were isolated in 65% of cases. When it was lower, isolation failed in 95% of the cases. According to our results, cardiac fluids appear to be a relevant matrix for toxoplasmosis survey in meat.

Identification of Trichinella isolates with random amplified polymorphic DNA markers.

Investigations using isoenzyme analysis have demonstrated genetic variation at the protein level between different isolates of Trichinella. La Rosa et al. (1992) recently reported 8 distinct gene pools obtained after analysis of 27 enzymes of 152 Trichinella isolates from 5 continents. Five of these patterns represent the five species described thus far: T. spiralis (T1), T. nativa (T2), T. pseudospiralis (T4), T. nelsoni (T7), and T. britovi (T3); Pozio et al. 1992). One drawback of this technique could be the number of enzymes required for analysis to distinguish the different species. DNA analysis has also been proposed to compare different isolates by means of restriction fragment length polymorphisms (RFLP; Klassen et al. 1986; Dick et al. 1990; Zarlenga et al. 1991) or polymerase chain reaction (Dupouy-Camet et al. 1991; Dick et al. 1992; Soul6 et al. 1993). However, these techniques, usefull in differentiating some isolates, could not identify the different species. Recent advances in genetic research have included the development of an adaptation of the polymerase chain reaction known as the random amplified polymorphic DNA polymerase chain reaction (RAPDPCR). The technique is fast, easy to perform, and requires a small amount of DNA and no prior sequence information (Williams et al. 1990). RAPD assay is based on the use of single synthetic 10-mer oligodeoxynucleotides as primers in a DNA-amplification reaction. The primer is short, increasing the probability that two priming sites occur in the genome close to each other in inverted orientation. Competition between many initial primer extension products and a low annealing temperature (36 ~ C) results in the synthesis of a few intense DNA bands after 4045 cycles of amplification. This

Cloning and analysis of a cDNA encoding a putative serine protease comprising two trypsin-like domains of Trichinella spiralis

The cDNA encoding a putative serine protease, TsSerP, was cloned by degenerative polymerase chain reaction and screening of the cDNA library from Trichinella spiralis adult–newborn larvae stage. Sequence analysis revealed the presence of two trypsin-like serine protease domains flanking a hydrophilic domain, with the catalytic triad residue histidine in the alpha domain substituted by an arginine residue. Southern blots indicated that this was a single copy gene in the parasite genome. Northern blots demonstrated a single 2.3-kb transcript during the muscle larvae and adult stages of T. spiralis. The recombinant protein from the TsSerP beta domain (βSerP) was produced but not recognised by T. spiralis-infected swine serum. An anti-βSerP polyclonal serum detected a 69-kDa polypeptide in the soluble antigens of T. spiralis muscle larvae. Immunolocalisation analysis located TsSerP on the inner layer of the cuticle and oesophagus of the parasite, suggesting a potential role in its moulting and/or digestive functions.

Animaux réservoirs de Toxoplasma gondii : état des lieux en France

Resume La toxoplasmose est une maladie largement distribuee a travers le monde. Elle est due a Toxoplasma gondii , un protozoaire ayant les felides comme hote definitif (le chat) et les vertebres homeothermes comme hotes intermediaires. L’infection humaine se fait principalement par voie orale, soit par l’ingestion d’oocystes excretes par voie fecale par les chats, soit par l’ingestion de kystes tissulaires qui peuvent etre presents dans une grande variete de produits carnes. La transmission directe a partir d’un chat a son proprietaire n’est probablement pas frequente, mais les populations de chats sont plutot a l’origine d’une contamination environnementale large et durable. En France, des plans de surveillance dans les viandes ovines, bovines et porcines, destinees a la consommation humaine ont permis de mettre en evidence une seroprevalence de Toxoplasma gondii qui se situe, selon l’espece et l’origine des viandes, entre 2,46% pour les porcelets hors-sol et 69,5% pour les ovins adultes d’origine francaise. Le role des animaux sauvages en tant qu’hotes intermediaires du parasite est egalement a prendre en compte dans l’epidemiologie de la toxoplasmose, principalement comme facteur de dispersion du parasite et, secondairement, comme agent de contamination humaine. En Guyane, l’hypothese de l’existence d’un cycle sauvage de Toxoplasma gondii est evoquee depuis quelques annees, mettant en jeu les felides sauvages et leurs proies, mammiferes et oiseaux, dont certaines especes peuvent etre des produits de chasse pour les humains.