ITRAQ-based proteomics of excretory-secretory products of Trichinella spiralis and Trichinella pseudospiralis at the muscle larva stage

The presence of Trichinella larvae was investigated in 247 samples taken from domestic, synanthropic and sylvatic animals, collected during 1996 to 2005 in 12 endemic provinces of Trichinella infection in Argentina. Muscle larvae of Trichinella from 65 infected animals were identified at the species level by single larva nested polymerase chain reaction (PCR) technique based on the variability within the expansion segment V (ESV) region of the ribosomal DNA. Trichinella infections were found in 97 of 164 pigs, 38 of 56 pork products, two domestic dogs, one domestic cat, 7 of 11 armadillos and 3 of 9 synanthropic rats. All Trichinella isolates were identified as Trichinella spiralis by nested PCR. These findings add new data on the epidemiology of trichinellosis and should be considered when implementing new strategies to control this zoonosis.

Objective To establish the ELISA method for detection of anti-Trichinella antibody IgG in sera and saliva. Methods The phalanx titration was used for the selection of the ELISA experimental conditions such as the optimal coating concentrations of T. spiralis antigens including muscle larval soluble antigen (MLSA),muscle larval excretory-secretory antigen (MLESA),adult wornl soluble antigen (AWSA),adult worm excretory-secretory antigen (AWESA), the dilutions of sera and saliva, the dilutions of the goat anti-rabbit and the goat anti-human IgG conjugated with horseradish peroxidase (HRP).Sera and saliva from 20 rabbits,10 patients infected with T. spiralis were used for the sensitivity assay of the 4 antigens.while sera and saliva from 20 healthy parasite-free rabbits and persons,sera and saliva from 38 rabbits and patients infected with other parasites were detected for the specificity of the 4 antigens.Results The optimal coating concentrations of the 4 antigens were 8.0 μg/ml,6.0 μg/ml,10.0 μg/ml,9.0 μg/ml,respectively.The optimal dilutions of sera were 1:100,1:200,1:50,1:200 respectively.while saliva was used without dilution.The dilutions of the goat anti.rabbit and the goat anti-human IgG were 1:2 500 and 1:2 000.respectively.The sensitivities of the 4 antigens for detecting the sera and saliva from rabbits infected with T. spiralis were 100% and 80%-100%.respectively.The sensitivities of the 4 antigens for detecting the sera and saliva from patients with trichinellosis were 100% and 60%-80%.respectively.The specificities of the 4 antigens for detecting the sera and saliva were 81.03%,89.65%,77.59%.82.76%and 93.10%,96.55%,89.65%.91.35%respectively. Conclusion The ELISA assays for detection of anti-T.spiralis IgG antibody in serum and saliva samples were established.Saliva samples were promising in substituting serum for detection of triehinellosis when there were diffieulties to collect serum samples. Key words: Trichinella spiralis; ELIsA; Serum; Saliva

The occurrence of Trichinella spiralis larvae in tissues other than skeletal muscles.

Experimental observation of the development of Trichinella spiralis muscle larvae in mice

Trichinellosis is a concern for public health aut horities in Europe and efforts have been made to promote and fund European networks Key scientists in this field have been identified and meet or communicate regularly to improve the management and prevention of this potentially lethal disease. Key preventive measures include training the technician s in charge of meat control and educating the consu mers to cook potentially infected meat thoroughly. Trichinellosis is a zoonotic disease caused by the ingestion of raw meat containing larvae of the nematode Trichinella . Four species of Trichinella are found in Europe : Trichinella spiralis (cosmopolitan), T. britovi (in wildlife from mountainous areas), T. nativa (in wildlife from colder and northern regions) and T. pseudospiralis (a cosmopolitan nonencapsulating species). Human trichinellosis causes high fever, facial oedema, myositis and eosinophilia. It can be a seri ous disease, particularly in elderly patients in wh om neurological or cardiovascular complications can le ad to death.

Trichinellosis is a cosmopolitan zoonosis due to a nematode threadworm, Trichinella, essentially Trichinella spiralis. Human cases mostly appeared sporadically, sometimes endemically, related with consumption of larva stinking meat. We report two cases of trichinellosis, including a myocarditis, caused by Trichinella britovi after consumption of frozen wild boar meat.

Trichinellosis is a food-borne parasitic zoonosis with a yearly incidence of about 10,000 clinical cases worldwide. It is one of the most serious zoonotic diseases in Romania with more than 28,000 human cases reported over the last 25 years. In this context Romania remains the country with the highest infestation with Trichinella from the word. The aim of the paper it is to identify the simple repetitive sequences from Trichinella genus through a biomolecular analysis of isolates. After conducting the DNA extraction from 50-100 muscle larvae of the six selective strains (jackal, wolf 1, wolf 4, 7.97, 7.42 and 7.29) using the “ QIAamp DNA” (Qiagen, Germany) the amplification with ESV primers (forward and reverse) using the “mi-Taq Mix Kit ”(Metabion, Germany) followed, obtaining in this manner a positive amplification for all the six strains taken into study, revealing also the method's efficiency in the biomolecular identification of Trichinella strains. The goal of this research, in perspective, is to improve the DNA extraction from one LM, in order to reduce the chemical and biological reagents. The positive amplification of the genomic DNA from one larva may have favorable repercussions on the genetic variability analysis in the population of Trichinella studied, which can be applied on various individuals belonging to the same population.

Recombinant protein was produced from the cDNA library of Trichinella pseudospiralis, which seemed to form part of the excretory-secretory (ES) products. The library was constructed from cDNA of muscle larvae at 1 month post-infection, and immunoscreened with antibody against T. pseudospiralis ES products. A clone, designated Tp21-3, contained a cDNA transcript of 657 bp in length with a single open reading frame, which encoded 172 amino acids (19617 Da in the estimated molecular mass). The predicted amino acid sequence of clone Tp21-3 had a similarity of 76% to that of clone ORF 17.20 (GenBank under accession number U88239) from T. spiralis. The recombinant fusion proteins encoded by clone Tp21-3 were produced in an Escherichia coli expression system and affinity purified. On Western blotting analysis, Tp21-3 recombinant proteins migrated at 40 kDa and reacted to antibody against T. pseudospiralis ES products and T. pseudospiralis-infected sera. Sera were developed against Tp 21-3 recombinant proteins, which reacted to a single band migrating at 21 kDa in crude worm extract and ES products from T. pseudospiralis on Western blotting analysis, and reacted with stichocytes of T. pseudospiralis on immunohistochemical staining.

Objective To analyze the protein components and the immunoreactivity of Trichinella spiralis new born larva (NBL) soluble antigens at different stages in Dali,Yunnan.Methods The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein components of the worm soluble antigens of NBL at different stages, and the Western-blot was used to analyze the immunoreactivity of the worm soluble antigens of NBL at different stages against T.spiralis infected rats.Results The results from SDS-PAGE analysis displayed that the worm soluble antigens of T.spiralis NBL on 24 h showed 36 protein bands with the range between Mr160000 and 10000,among which the 8 main bands were Mr73000,64000,58000,54000,46000,40000,34000 and 25000,respectively.The worm soluble antigens of T.spiralis NBL on 48 h displayed that 40 proteins bands with the range between 172000 and 10000,among which the 17 main bands were 120000,102000,89000,87000,79000,74000,72000,64000,58000,54000,46000,40000,34000,32000,25000,18000 and 10000,respectively.The results of Western-blot showed that 7 protein bands of the worm soluble antigens from T.spiralis NBL on 24 h reacted with the serum of rat anti-T.spiralis NBL.Whereas,14 protein bands of the worm soluble antigens from T.spiralis NBL on 48 h reacted with the serum of rat anti-T.spiralis NBL.Moreover,the negative serum had no cross-reactivity to corresponding proteins.Conclusion The worm soluble antigens of T.spiralis NBL on 24 h had 7 same protein components in comparison with NBL on 48 h.The protein components from the worm antigens of T.spiralis NBL increased during the development of the worm.Western-blot displayed that the serum of rat anti-T.spiralis NBL had stronger specific recognition to the 6 protein components from the worm soluble antigen of T.spiralis NBL both on 24 h and 48 h.Thus,these protein components had stronger immunoreactivity. Key words: Ttichinella spiralis; New born larvae; Worm soluble antigens; Protein components; Immunoreactivity

A parasitologic study is presented of 103 adult house rats from various sites in San Jose, Costa Rica. Seven specimens were Rattus rattus rattus, and the rest were Rattus norvegicus. The following protozoa were found in trophic form when feces were examined for eggs and larvae of helminths: Trichomonas, Giardia, Hexamita, Chilomastix, and Entamoeba. Trypanosoma lewisi was found in the blood of 7 out of 17 rats examined (8,8%) The following nematodes and cestodes were found with the incidence indicated: Strongyloides ratti (41,7 %); Aspiculuris tetraptera (1,9 %); Nipostron­gylus brasiliensis (47,6%); Gongylonema neoplasticum (0,9 %); Protospirura muris (8,7%); Trichosomoides crassicauda (23,3%); Taenia taeniformis larvae (33%); Hymenolepis diminuta (43,6%); and H. nana (0,9 %). No larvae of Trichinella spiralis were found in diaphragms examined. The acanthocephalian Moniliformis moniliformis was found in 18,4 per cent of the rats. Arthropods found as ectoparasites were, in arder of incidence, Echino­laelaps echidninus (14,5%), Xenopsilla cheopis (7,7 %) and Ctenopsyllus se­gnis (1,9%). These percent data should be regarded as minimum figures, as only 61 rats were brought alive to the laboratory.

Objective To detect Trichinella spiralis DNA by loop-mediated isothermal amplification (LAMP).Methods DNA was extracted by phenol-chloroform extraction method from Trichinella larva.Four primers were designed based on 18S rRNA sequence and used for LAMP assay.To evaluate the specificity of the LAMP method,DNA sample derived from Schistosoma japonicum was used as control.For evaluating the sensitivity of LAMP,the genomic DNA derived from T.spiralis was serially diluted and amplified by LAMP.LAMP reaction mixture was stained with SYBR green I dye and the result was determined according to the color developed in the tube by the naked eye.Green indicated positive reaction while brown was negative.Results After LAMP reaction,the test tube which used genomic DNA derived from T.spiralis appeared to be green,while control was brown.The sensitivity of LAMP assay in this study reached up to 415 fg/μl.Conclusion LAMP assay was established for the detection of DNA sample of T.spiralis. Key words: Trichinella spiralis ; Loop-mediated isothermal amplification ; Detection ;

Objective To predict the antigenicity of two new Trichinella genes,Ad48 h-Ts3 and tetraspanin,synthesize their gene sequences,express their recombinant proteins and evaluate their potential for diagnosis.Methods The potential antigenicity of the two proteins,tetraspanin family protein and adult stage protein Ad48 h-Ts3 were determined according to the result of their hydrophilicity and hydrophobicity predictions.The sequences of these two genes were synthesized after codon optimization and were subcloned into pET28a expression vector and expressed in E.coli system.Western blotting was performed to evaluate the diagnosis value of the proteins using mouse infected sera.Results Bioninformatic analysis showed that tetraspanin gene of Trichinella spiralis,which bears two transmembrane domains,has homology gene in other species.However,Ad48 h-Ts3 gene,which has plenty of antigenic epitopes,is unique in Trichinella spiralis.Genes were subcloned into expression vector and recombinant Ad48 h-Ts3 protein was expressed successfully in E.coli system while no obvious expression was observed in tetraspaning expression.Western blotting showed the expressive recombinant Ad48 hTs3 protein could be recognized by Trichinella infected sera obtained from mice.Conclusion The adult stage protein,Ad48 h-Ts3 protein was expressed successfully in E.coli and had a potential value for diagnosis. Key words: Trichinella spiralis; Diagnosis; Tetraspain; Adult stage protein

Trichinellosis caused by the Nematode Trichinella is azoonotic disease which occurs worldwide and affects abroad range of different species including mammals, birds and amphibians. Trichinella spiralis is found in production animals (pigs, horses) in temperate climate zones and can also be found in animals in close contact with these production animals (e.g. dogs, cats, rats). The PrioCHECK® Trichinella Ab is a reliable and fast diagnostic test for detection of antibodies against Trichinella in porcine serum and meat juice samples, and can be used for monitoring and surveillance purposes. Keywords: Trichinella, pig, diagnosis, ELISA, Saranda.

Objective To study the levels of Th1 and Th2 type cytokines in 3-nitrobenzene sulfonate(TNBS) and oxazolone(OXZ) colitis without or with Trichinella spiralis( T. spiralis) infection. Methods Female BALB/c mice were randomly divided into 4 groups: 50% Ethanol, T. spiralis only, TNBS or OXZ,T. spiralis +TNBS or OXZ(at least 6 in each group when mice were killed). The levels of IFN-γ, IL-12,IL-4, IL-10 in colon in mice with colitis induced by TNBS or OXZ without or with T. spiralis infection 3 d and 7 d post-induction were assessed using ELISA method. Results Colonic protein levels of IFN-γand IL-12 were significantly increased 3 d and 7 d after intra-colonic injection of TNBS( P ＜0.05 ). Concurrent infection with T. spiralis prevented this rise in IFN-γand IL-12 secretion and tended to induce a rise in colonic IL-4 and IL-10 content(P ＜0. 05). The levels of IFN-γ, IL-4 and IL-10 protein in colitic mice colon with prior nematode infection on days 3 and 7 post-induction of colitis were significantly higher than that seen in colitic mice without prior nematode infection ( P ＜ 0.05 ). Conclusion T. spiralis infection significantly attenuates TNBS-induced colitis in mice. The local immunologic mechanism is that T. spiralis can down-regulate strongly Th1-type immune response of colitis and up-regulate Th2 response, Tr1-cytokines. According to the change of Th1/Th2 cytokines, prior T. spiralis infection doesn't reduce the severity of OXZ-induced colitis, but without aggravating colitis. The exactly immunologic mechanism deserve to be explored deeply. Key words: Trichinella spiralis; Inflammatory bowel disease

Trichinosis is one of the most common zoonotic parasitic diseases,a kind of important foodborne parasitic disease with serious harm to human health.Dendritic cells are a kind of innate immune cells.As one of the most important antigen-presenting cells in the intestinal mucosal immune system of the host,it is closely related to the intestinal immune system.In recent years,its effect on parasite infection and host immune responses,especially in the Trichinella spiralis infection and immunity has attracted people's attention.In this review,the research progress at home and abroad on the host intestinal mucosal dendritic cells involved in a series of immunological reactions and roles in Trichinella infection was reviewed. Key words: Trichinella spiralis; Dendritic cells; Immunity; Intestinal mucosa