A Trichinella spiralis new born larvae-specific protein, Ts-NBL1, interacts with host's cell vimentin.

The parasitic nematode Trichinella has a special relationship with its host as it has a unique intracellular location within the feeder cell which is a structure derived from skeletal muscle fiber. It has been proposed that "parakines" secreted by Trichinella larvae serve as messengers to implement communication between the parasite and the muscle cells through a molecular cross-talk to ensure permanent coexistence within the host. The Ts-NBL1 protein is considered to be a potential key "parakine" involved in the early invasion of the muscle fiber and its transformation into a feeder cell during Trichinella spiralis infection. This study used for the first time yeast two-hybrid (Y2H) technology in Trichinella to identify Ts-NBL1 interacting proteins. GST co-affinity purification experiments confirmed vimentin as an important interactor. The discovery of the new host proteins interacting with Ts-NBL1 will help to suggest that Ts-NBL1 contributes to participate in the capsule formation of feeder cells and provide ideas for understanding the molecular and cellular mechanisms involved in the survival of Trichinella in the host.

Seroprevalance of Trichinella Spp. in Wild Boars (Sus Scrofa) from Bihor County, Western Romania.

The wild boar (Sus scrofa) has a wide geographical distribution and can be an important source of Trichinella spp. infection in humans in Romania. The objective of this study was to identify the presence of Trichinella spp. in the wild boar population in Bihor County, Romania. Eighty four plasma and diaphragm samples, collected from wild boars, were included in this study. Artificial digestion, ELISA and Western blot were performed on these specimens. All diaphragm samples were negative for Trichinella larvae in artificial digestion, while in ELISA, 54 (64.2 %) plasma samples were positive and 6 (7.1 %) plasma samples were doubtful. Western blot was performed on 26 plasma samples from which only 6 (23.0 %) gave a positive result. Serological evidences indicate the presence of Trichinella spp. in wild boars from western Romania. Therefore, human consumers might be at risk to ingest Trichinella larvae, even in low numbers.

Influence of adjuvant formulation on inducing immune response in mice immunized with a recombinant serpin from Trichinella spiralis.

Nematodes of the genus Trichinella are one of the most widespread zoonotic pathogens on the world, and they can still cause major public health problems in many parts of the world. Vaccination against the helminth nematode Trichinella could be a good strategy to reduce the risk of human and animal infection. It was our aim to evaluate three adjuvants, which could be used as an efficient vaccine for animals in combination with rTs-Serpin antigen. In this study, BALB/c mice were vaccinated by an intramuscular route with rTs-Serpin antigen from the parasite Trichinella spiralis in combination with three different adjuvant formulations: Montanide ISA201, Montanide IMS 1313 N PR VG and Freund's complete adjuvant/Freund's incomplete adjuvant (FCA/FIA). The dynamics of IgG, IgM, IgE and cytokine production from spleen cells and worm reduction rate of the vaccinated mice were analysed. The results showed that rTs-serpin can induce partial protection against Trichinella larvae challenge in mice, when compared to the FCA-/FIA-formulated vaccination, the IMS1313 plus rTs-serpin mixture showed higher humoral immunity and similar levels of cellular immunity and worm reduction rate. The study suggested that Montanide IMS nanoparticles 1313 are as effective as FCA but less toxic; thus, Montanide IMS nanoparticles 1313 can be used as a good candidate of adjuvant for developing vaccine against Trichinella spiralis.

Developmental profile of select immune cells in mice infected with Trichinella spiralis during the intestinal phase.

Trichinella spiralis can cause immunosuppression during the intestinal phase of early infection. However, changes in the peripheral blood during T. spiralis early infection remain unclear. Here, select immune cells in mice infected with 500 muscle larvae (ML) of T. spiralis during the intestinal phase of infection were studied. First, the recovery rates of the intestinal worms and female fecundity were determined, and the results showed that the intestinal worms were completely eliminated at 17 days post-infection (dpi) and that large numbers of new-born larvae (NBL) were generated from 5 to 9dpi. Using flow cytometry, it was shown that the number of CD4+ T cells and CD8+ T cells increased over the entire intestinal phase, except on 7dpi when CD4+ T cells decreased significantly compared to the control groups. Although both CD4+ and CD8+ T cells increased, CD8+ T cells increased more than CD4+ T cells, leading to a lower CD4+/CD8+ ratio compared to the control group. Subsequently, a depression of the proliferative response of T cells to concanavalin A (Con A) was noticed at 7 and 11dpi. Although the proliferative response of B cells to LPS was enhanced, the number of B cells from mouse peripheral blood stimulated by T. spiralis antigens showed no differences with the control group prior to 11dpi. The expression of CD14 on monocyte-macrophages decreased during the same period, which meant that the antigen-presenting response was reduced in the immune system of the infected mice. Moreover, the alternatively activated macrophages were induced in T. spiralis early infection. These data provide a better understanding of the development of the intestinal immune response in mice infected with T. spiralis.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts.

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell-cell interactions during T. spiralis infection.

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'.

Identification and characterization of immunodominant linear epitopes on the antigenic region of a serine protease in newborn Trichinella larvae.

An immunodominant serine protease of Trichinella spiralis named NBL1 showed encouraging potential in early diagnosis of trichinellosis in pigs and elicited protective immune responses during infection of animals. To further define serological reagents for diagnostic use, the specific epitopes on NBL protein recognized by the antibody responses of different susceptible hosts need to be defined. The present study described comprehensive mapping of immunodominant linear epitopes in the antigenic region (NBL-C, the C-terminal part of the protein) using various serum samples obtained from three kinds of hosts - pig, wild boar and mice. We identified six peptides which were commonly recognized by sera from pigs experimentally infected with Trichinella and pigs immunized with rNBL1-C; five and four peptides were recognized by sera from wild boars and mice infected with Trichinella, respectively. Three peptides (NBL1-6, -7 and -9) were commonly recognized by antisera in all three hosts, which share the sequence PSSGSRPTYP. We also found that one peptide (NBL1-12) was only recognized by antibodies from pigs immunized with rNBL1-C. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of subunit vaccines and diagnostic tools for trichinellosis.

Other vector-borne parasitic diseases: animal helminthiases, bovine besnoitiosis and malaria.

The parasitic diseases discussed elsewhere in this issue of the Scientific and Technical Review are not the only ones to make use of biological vectors (such as mosquitoes or ticks) or mechanical vectors (such as horse flies or Stomoxys flies). The authors discuss two major groups of vector-borne parasitic diseases: firstly, helminthiasis, along with animal filariasis and onchocerciasis, which are parasitic diseases that often take a heavytoll on artiodactylsthroughoutthe world; secondly, parasitic diseases caused by vector-borne protists, foremost of which is bovine besnoitiosis (or anasarca of cattle), which has recently spread through Europe by a dual mode of transmission (direct and by vector). Other protists, such as Plasmodium and Hepatozoon, are also described briefly.

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.

An anti-tumor protein produced by Trichinella spiralis induces apoptosis in human hepatoma H7402 cells.

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic myeloid leukemia cell line (K562) and a human hepatoma cell line (H7402) using the display library. The protein encoded by the A200711 gene was identified and analyzed using protein analysis software. To test the antitumor effects of A200711, variations in cell proliferation and apoptosis were monitored after recombinant pEGFP-N1-A200711 was transfected into H7402 cells. The results show that the expressed target gene successfully induced apoptosis in H7402 cells as measured by Hoechst-PI staining, MTT assay (p<0.05). This study warrants further investigation into the therapeutic use of A200711 for anti-hepatocellular carcinomas.

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.

Modulation of inflammatory bowel disease in a mouse model following infection with Trichinella spiralis.

Infection of mice with Trichinella spiralis redirects the mucosal immune system from a Th1 to a protective Th2 response with a reduction in the severity of trinitrobenzesulfonic acid-induced colonic damage. T. spiralis infection induced IL-10 production in a dose-dependent manner in oxazolone (OXZ)-induced colitis. This phenomenon may be responsible for the lack of efficacy of T. spiralis in the treatment of OXZ-induced colitis. These results indicate that if the source of increased IL-10 production is identified and addressed, T. spiralis may alter the Th2 response.

New data in France on the trematode Alaria alata (Goeze, 1792) obtained during Trichinella inspections.

The trematode Alaria alata is a cosmopolite parasite found in red foxes (Vulpes vulpes), the main definitive host in Europe. In contrast only few data are reported in wild boars (Sus scrofa), a paratenic host. The aim of this paper is to describe the importance and distribution of Alaria alata mesocercariae in wild boars, information is given by findings of these larvae during Trichinella mandatory meat inspection on wild boars' carcasses aimed for human consumption. More than a hundred cases of mesocercariae positive animals are found every year in the East of France. First investigations on the parasite's resistance to deep-freezing in meat are presented in this work.

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.

Epidemiological survey of trichinellosis in wild boar (Sus scrofa) and fox (Vulpes vulpes) in a French insular region, Corsica.

The Mediterranean island of Corsica was considered Trichinella-free until 2004, when T. britovi larvae were discovered in domestic pigs at meat inspection. One red fox was also found infected the same year and in the same area than the infected pigs. This last finding highlighted the presence of trichinellosis in Corsican wildlife. A Trichinella survey was thus performed in wild boar (Sus scrofa) and fox (Vulpes vulpes), the two large wild species present on the island, to determine prevalence of muscle larvae and antibodies. Diaphragm muscles of 1881 wild boars and 74 forelegs of foxes were tested by artificial digestion. No Trichinella larva was identified. The highly sensitive ELISA was used to test muscle fluid samples of 1492 wild boars. The apparent serological prevalence of Trichinella infections in wild boar was 2.01% (95% CI: 1.36-2.86). The present results suggest that wildlife is currently exposed to Trichinella in Corsica. In this context, adequate cooking and veterinary controls of meat offer the only complete sanitary warranties to consumers.

Seroprevalence and factors associated with Toxoplasma gondii infection in wild boar ( Sus scrofa) in a Mediterranean island.

Knowledge of the factors affecting the presence of Toxoplasma gondii in wildlife is limited. Here we analyse which local landscape characteristics are associated with the presence of toxoplasmosis in wild boar, Sus scrofa, on the island of Corsica, France. Meat juice samples from 1399 wild boars collected during two hunting seasons were tested for T. gondii antibodies using the modified agglutination test (titre 1:4). The overall seroprevalence was 0.55 (95% CI 0.50-0.59) for the first year and 0.33 (95% CI 0.29-0.35) for the second year. Seroprevalence varied according to age and county. At the county level, seropositivity in adults was related to farm density during year 1, and to habitat fragmentation, farm density and altitude during year 2. The exposure of wild boar to T. gondii is thus variable according to landscape characteristics and probably results in a variable risk of transmission of toxoplasmosis to humans.

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs.

Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA.

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.

Molecular cloning and characterization of heat shock protein 70 from Trichinella spiralis.

A cDNA encoding heat shock protein 70 of Trichinella spiralis (Ts-Hsp70) was identified by immunoscreening the adult T. spiralis cDNA library with rabbit antisera against T. spiralis adult extracts. The open reading frame of Ts-Hsp70 cDNA encoded a 623-amino acid peptide with a predicted molecular weight of 68.7kDa, which shares a high degree of sequence conservation with HSP70s from other parasites. Recombinant Ts-Hsp70 was expressed in Escherichia coli and purified with nickel column chromatography. Western blot analysis showed that recombinant Ts-Hsp70 could be recognized not only by trichinellosis patient sera, but also by T. spiralis-infected sera from rabbits, swine, and mice. Mice vaccinated with recombinant Ts-Hsp70 formulated with Freund's adjuvant exhibited strong humoral immune responses indicated by high titer of IgG antibody and significant muscle larval reduction (37%) after being challenged with T. spiralis larvae. The present results indicate that Ts-Hsp70 is a possible candidate vaccine against T. spiralis infection.