Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Factors associated with Toxoplasma gondii infection in confined farrow-to-finish pig herds in western France: an exploratory study in 60 herds.

BACKGROUND: Infection by Toxoplasma gondii postnatally can occur after ingestion of contaminated meat or water (tissue cysts/oocysts). In Europe, percentage of meat borne infections is estimated between 30 and 63 %, out of which pork makes the most important source. The aim of this study was to (i) investigate the seroprevalence of T. gondii in intensive pig farms from western France; and (ii) identify the risk factors associated with seropositivity. METHODS: Data were collected between November 2006 and February 2008 in 60 intensive farrow-to-finish farms, where sera were taken from 3595 fattening pigs, weaned and suckling piglets. Information about three classes of potential seropositivity risk factors were obtained through a questionnaire concerning: (i) breeding characteristics; (ii) farm management; and (iii) husbandry and hygiene. The modified agglutination test (MAT) was used for detection of specific anti T. gondii antibodies in pig sera, starting from 1/6 dilution. RESULTS: The overall proportion of seropositive animals was 6.9 %, but the proportion of herds with at least one positive pig was 100 %. Multivariate logistic mixed model showed an increased seropositivity risk in weaned compared to suckling piglets, and a decreasing risk for mid-sized and large farms. The presence of a Danish entry facility, that clearly separates clean and dirty areas, had a protective effect on T. gondii seropositivity as well. CONCLUSIONS: The observed proportion of herds with at least one T. gondii seropositive animal provides further evidence that even in confined conditions of pig breeding, infection occurs, and is common. The highest risk for acquiring T. gondii is at the end of weaning period. Smaller confined pig farms demonstrate higher T. gondii seropositivity levels. This study also showed that Danish entry on farm buildings provides effective protection against T. gondii.

Toxoplasma gondii infection in pork produced in France.

The aim of this study was to assess the seroprevalence of the Toxoplasma gondii parasite in pork produced in France, and to determine infection risk factors. An innovative survey was designed based on annual numbers of slaughtered pigs from intensive and outdoor farms in France. A total of 1549 samples of cardiac fluids were collected from pig hearts to determine seroprevalence using a Modified Agglutination Test. Of those, 160 hearts were bio-assayed in mice to isolate live parasites. The overall seroprevalence among fattening pigs was 2·9%. The adjusted seroprevalence in pigs from intensive farms was 3·0%; the highest in sows (13·4%); 2·9% in fattening pigs and 2·6% in piglets. Adjusted seroprevalence in fattening animals from outdoor farms was 6·3%. Strains were isolated from 41 animals and all were genotyped by Restriction Fragment Length Polymorphism as type II. Risk-factor analysis showed that the risk of infection was more than three times higher for outdoor pigs, and that sows' risk was almost five times higher than that of fattening animals. This study provides further evidence of extensive pork infection with T. gondii regardless of breeding systems, indicating that farm conditions are still insufficient to guarantee 'Toxoplasma-free pork'.

Major Parasitic Zoonoses Associated with Dogs and Cats in Europe.

Some of the most important zoonotic infectious diseases are associated with parasites transmitted from companion animals to man. This review describes the main parasitic zoonoses in Europe related to dogs and cats, with particular emphasis on their current epidemiology. Toxoplasmosis, leishmaniosis, giardiosis, echinococcosis, dirofilariosis and toxocariosis are described from the animal, as well as from the human host perspectives, with an emphasis on parasite life cycle, transmission, pathogenicity, prevention and identification of knowledge gaps. In addition, priorities for research and intervention in order to decrease the risks and burden of these diseases are presented. Preventing zoonotic parasitic infections requires an integrated multidisciplinary 'One Health' approach involving collaboration between veterinary and medical scientists, policy makers and public health officials.

[Serological survey of animal toxoplasmosis in Senegal]. / Enquête de séroprévalence de la toxoplasmose animale au Sénégal.

Toxoplasma gondii is an obligate, intracellular, parasitic protozoan within the phylum Apicomplexa that causes toxoplasmosis in mammalian hosts (including humans) and birds. We used modified direct agglutination test for the screening of the animals' sera collected in Senegal. In total, 419 animals' sera have been studied: 103 bovines, 43 sheep, 52 goats, 63 horses, 13 donkeys and 145 dogs. The collection of sera was performed in four different regions of Senegal: Dakar, Sine Saloum, Kedougou and Basse Casamance from 2011 to 2013. We have revealed antibodies in 13% of bovines, 16% of sheep, 15% of goats, 30% of horses, 23% of donkeys and 67% of dogs. Private dogs from villages were more often to have the anti-Toxoplasma antibodies compared to security society-owned dogs from Dakar. It may be explained by different meal consumed by dogs (factory-produced meal for dogs from Dakar vs. irregular sources for village dogs). Intense circulation of T. gondii in the studied zone may explain the unusually high seroprevalence among horses and donkeys. Tropical climate with high temperature and humidity is favorable for the conservation of oocysts of T. gondii. Results presented here may contribute to the evaluation of the risks of toxoplasmosis in humans in Senegal.

Detection of anti-Trichinella antibodies in serum of experimentally-infected swine by immunochromatographic strip.

An immunochromatographic strip method, developed with the excretory-secretory antigens from muscle larvae (ML) of Trichinella spiralis labeled with colloidal gold, was used for the detection of anti-Trichinella antibodies in serum of experimentally-infected swine. Sera from swine infected with 200, 2000 and 20,000 infective ML were collected at different days post infection (dpi) and used to evaluate the method. The strip method was shown able to detect anti-Trichinella antibodies by 35 dpi, 28 dpi and 21 dpi for the three different infection doses, respectively, and closely correlated with the results of an ELISA test. The strip method is rapid and easy to perform and is suggested as an acceptable alternative for clinical laboratories lacking specialized equipment, and for field diagnosis of trichinellosis.

Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection.

The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time course bleeds coinciding with early worm development. Seventeen positive clones were isolated using serum obtained at 20 days post infection (dpi). All clones corresponded to one gene that exhibited high sequence identity with the T. spiralis ATP-dependent RNA helicase DDX19B which is involved in parasite growth and development. In addition, nine additional positive clones representing 5 unique genes were identified when the library was screened with 30 dpi serum; four of these five genes displayed high similarity with members of a putative T. spiralis serine protease family known to be involved in host invasion and host-parasite interactions. The remaining gene aligned with the T. spiralis hypothetical ORF 11.30. The identification of these antigens provides potential candidates for the early diagnosis of trichinellosis and for the development of a vaccine against this parasite.

Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis.

Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections.

Use of mitochondrial RNA genes for the differentiation of four Trichinella species by multiplex PCR amplification.

Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species.

Species identification of Trichinella isolates from China.

Two species of Trichinella were identified from China by means of PCR amplification of the mitochondrial small subunit ribosomal DNA and the expansion segment V region of the ribosomal DNA. Seven isolates originating from domestic pig and one isolate originating from dog showed identical DNA banding pattern to Trichinella spiralis, and two isolates from dog showed DNA banding pattern identical to Trichinella nativa. Sequence analysis of the 5S rDNA inter-gene spacer region from the ten Trichinella isolates confirmed the existence of only two Trichinella species and revealed the inner species genetic variation within T. spiralis and T. nativa.

Animal Trichinella infection in Romania: geographical heterogeneity for the last 8 years.

Previous studies in southeastern Europe reported a high incidence of human trichinellosis and a high prevalence of Trichinella infection in animals in countries like Bulgaria, Croatia, Romania and Serbia. The aim of this study was, using routine Trichinella test data in pig and game animals, to investigate the extent of Trichinella infection in slaughtered animals in Romania, over the period of 1997-2004, and to identify possible differences in prevalence among the various regions of Romania. Trichinella infection data were obtained from trichinelloscopic examinations of domestic (backyard and industrial reared pigs) and game species (wild boar and bears). A marked difference between Transylvania and other counties of Romania, observed for human trichinellosis, was taken into account when analyzing Trichinella epidemiological data. A cumulative prevalence of 8 cases/10(4) animals tested for pigs, 9 cases/10(3) animals tested for wild boars, and 13.1 cases/10(2) animals tested for bears was obtained for the 8 years period. Analysis of animal prevalence data demonstrated a geographical heterogeneity: whereas Trichinella prevalence in pigs is much lower in Transylvania than in the other counties, Trichinella prevalence in game animals is similar for the different regions. This observation suggests that, in Romania, rather than the levels of the parasite circulating in domestic and game animals, it was changes in the social and political structure of Romania in the 1990s, combined with inadequate meat inspection practices that were the main contributors to these geographic variations.

First identification of Trichinella sp. in golden jackal (Canis aureus) in Romania.

Larvae of Trichinella sp. were identified in a golden jackal (Canis aureus) from Romania by both trichinelloscopy and artificial digestion. The larvae were identified as Trichinella britovi using a multiplex polymerase chain reaction biotyping method. This is the first report of Trichinella sp. in a jackal in Romania.