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.

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.

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.

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.

Effects of conidia of various Aspergillus species on apoptosis of human pneumocytes and bronchial epithelial cells.

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species.

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.

[Toxoplasmosis: a survey on meat products in Côte d'Ivoire]. / Toxoplasmose: à propos d'une enquête sur la viande en Côte d'Ivoire.

Toxoplasmosis is a worldwide parasitic zoonosis that can cause severe problems under certain circumstances. Before the advent of the last-generation anti-retroviral drugs, estimation predicted that 50% of HIV-infected patients would develop toxoplasmosis (mainly central nervous system forms). It is the first clinical manifestation of AIDS in 20% of patients. This report describes an epidemiological survey on the seroprevalence of Toxoplasma antibodies in bushmeat and pork in the Côte d'Ivoire. The purpose was to determine how the parasite circulates among wild and domestic animals and to evaluate the risk of transmission to humans after ingestion of these meats. Fifteen samples of bushmeat were purchased on markets in 6 different cities. A total of 91 single samples of fresh pork raised at three different modern breeding facilities were collected from a slaughterhouse in Abidjan. Serological testing was performed on muscle fluid using an ELISA test (Pourquier Toxoplasma kit). No bushmeat sample was positive. Global seroprevalence in pork samples was 8.8% [range, 8.2-9.37]. The seroprevalence of toxoplasmosis measured in pork samples produced at modern livestock breeding facilities was lower than values reported in samples produced by traditional breeding in Africa. This finding suggests that the use of modern techniques excluding rodents (good hygiene) can reduce animal contamination. Curing (heat and smoking) may account for the absence of Toxoplasma antibodies in bushmeat. Public information campaigns concerning the risk of consuming meat containing cysts as well as raw vegetables contaminated with oocysts are needed to prevent transmission of toxoplasmosis.

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis.

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.

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.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures. This paper presents an overview of the current methods for Trichinella meat inspection and their implementation in the EU, listing advantages and disadvantages for each method, including some suggestions for specific points of improvement.

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.

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.

Experimental studies in pigs on Trichinella detection in different diagnostic matrices.

A total of 72 specific pathogen-free (SPF) and Iberian pigs (three animals per group) were inoculated with 200, 1000 or 20,000 muscle larvae of T. spiralis, T. nativa, T. britovi and T. pseudospiralis. For each animal, the muscle larva burden was evaluated in nine muscle samples by digestion. The anti-Trichinella IgG kinetics in blood samples, taken twice prior and at days 5, 10, 15, 20, 25, 30, 40, 50 and 60 post-inoculation, and in muscle juice, obtained at necropsy, was evaluated by an ELISA using an excretory/secretory antigen. The mean larval recovery rate in SPF/Iberian pigs corresponded with the level of inoculum dose, and tongue, diaphragm and masseter were identified as predilection muscles. In SPF and Iberian pigs receiving 20,000 larvae of T. spiralis, an earlier seroconversion was detected from day 25 post-inoculation. At a 10-fold dilution, the muscle juice showed a good test agreement with blood serum.