Trichinella britovi recombinant proteins produced in Pichia pastoris expression system for specific IgG antibody detection in the sera of mice and pigs infected with Trichinella spp.

Trichinellosis, a disease caused by infection with Trichinella spp, poses an economic problem in the animal sector and a recurrent health problem for humans. Discovering the new diagnostic tests may be achieved by identification and production of species- and stage-specific recombinant proteins of Trichinella genus which are recognized by the host antibodies after infection. In this study the T. britovi proteins identified earlier in excretory-secretory (ES) products: CTRL, ES21 and HSP20, were cloned and produced using a eukaryotic Pichia pastoris system. Their immunodiagnostic properties were verified by measuring the abundance of specific IgG antibodies in sera from mice and pigs experimentally infected with T. britovi or T. spiralis. The rTbCTRL and the rTbES21 proteins were more effectively produced and stable than rTbHSP20. The most sensitive protein for serodiagnostic purposes occurred to be CTRL; anti-rTbCTRL IgG level increased at 41 days post infection (dpi) in pigs infected with T. britovi and 45 dpi for those infected with T. spiralis. The rTbES21 protein was the most specific for the T. britovi species, as no antibody titers were observed in pigs infected with T. spiralis. Following the multiple-antigen strategy, the combination of rTbCTRL + rTbES21 was applied in ELISA, but no significant difference in IgG level was detected in the tested conditions.

Insight into Trichinella britovi Infection in Pigs: Effect of Various Infectious Doses on Larvae Density and Spatial Larvae Distribution in Carcasses and Comparison of the Detection of Anti-T. britovi IgG of Three Different Commercial ELISA Tests and Immunoblot Assay.

There is limited information available on the Trichinella britovi (T. britovi) muscle larvae (ML) distribution in pig muscle and the humoral immune response of pigs infected with moderate doses of this parasite; therefore, this study investigated the infectivity of a Polish strain of T. britovi for pigs, the antibody response of this host to various doses of T. britovi, and the efficiency of three different commercial ELISA kits and an immunoblot assay at detecting anti-T. britovi IgG. No significant differences in terms of the infection level of particular muscles or of whole carcasses between pigs infected with 3000 and those infected with 5000 ML of T. britovi were observed. The highest intensity of T. britovi infection was reported in the diaphragm pillars. The larvae of T. britovi showed homogeneous distribution with respect to the muscle side. Statistically, specific IgG antibodies against excretory-secretory (ES) antigens of Trichinella ML were first detected by all ELISA protocols on day 36 post infection; however, individual pig results showed some differences between the three tests applied. A significant increase in the level of anti-T. britovi IgG was observed between days 36 and 41 post infection, and from day 45 until day 62 after T. britovi infection, production of these antibodies reached its plateau phase. No positive correlation was found between the anti-T. britovi IgG level and the larvae density in 15 different muscles. Sera of T. britovi-infected pigs showed reactivity with T. britovi ML ES antigens of 62, 55, and 52 kDa. The results provide novel information on spatial larvae distribution in muscles and the humoral immune response of pigs exposed to two different doses of a Polish strain of T. britovi, extend knowledge on serological diagnostic tools which may be introduced in veterinary practice for the detection of T. britovi infections in pig production, and offer practical solutions for meat hygiene inspectors in the field at sampling sites when examining pig carcasses for Trichinella.

Sarcocystis cruzi infection in free-living European bison (Bison bonasus bonasus L.) from the Bialowieza Forest, Poland - A molecular analysis based on the cox1 gene.

European bison are susceptible to a range of pathogens which may influence their health, and hence, to ensure their protection, it is essential to provide effective monitoring of potential exposure. This study presents the first molecular confirmation of Sarcocystis cruzi infection in European bison based on PCR amplification of the cytochrome c oxidase subunit I (cox1) gene. A sample of heart tissue taken from one fifteen-year-old European bison cow was examined by light microscopy for the presence of heart sarcocysts. The genomic DNA isolated from any identified sarcocysts was subjected to PCR to amplify cox1 gene sequences, and the obtained amplicons were sequenced by Sanger dideoxy sequencing. Two partial cox1 sequences were obtained; they were identified as S. cruzi and deposited in the GenBank™ database under the accession numbers MW490605 and MW490606. BLAST analysis found them to demonstrate the closest similarity to S. levinei (MH255771-MH255779 and KU247874-KU247884), sharing an identity of 93.14-93.8 %. This is the first report to identify sarcocysts isolated from heart tissue of infected European bison living in the Bialowieza forest to species level using cox1 analysis. Our findings confirm that the European bison is a natural intermediate host for S. cruzi. As such, coordinators of future conservation programmes should consider the impact of these diseases on reintroduced animals.

Regulation of human THP-1 macrophage polarization by Trichinella spiralis.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1ß and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1ß and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1ß, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

Use of meat juice from racoons (Procyon lotor) collected from Central Europe for immunological detection of Trichinella spp.

The raccoon (Procyon lotor) is a species native to North America, but which is now spreading throughout Europe. Raccoons have been found to host various Trichinella species. The aim of the present study was to evaluate the effectiveness of using immunological testing of meat juice for determining the occurrence of Trichinella in raccoons. The studies were carried out on 139 animals from three European countries: the Czech Republic, Germany and Poland. Seven meat juice samples were found to be positive for antibodies to Trichinella by ELISA, and another seven were unclear. The ELISA results were confirmed by immunoblot: anti-Trichinella antibodies were identified in 9.35 % of the examined animals. Slight agreement (κ = 0.13) was found between the digestion method and the combined ELISA and immunoblot approach. From the results of our study, we concluded that meat juice may be used as a simple and convenient sample for detection of anti-Trichinella in racoons.

The first analysis of Trichinella spiralis and Trichinella britovi adult worm excretory-secretory proteins by two-dimensional electrophoresis coupled with LC-MS/MS.

The two most common Trichinella species present in European countries are Trichinella spiralis and Trichinella britovi. The consumption of raw or undercooked meat with invasive larvae results in trichinellosis. Currently, the most commonly used sources for detecting specific anti-Trichinella antibodies is ELISA with the muscle larvae (ML) excretory-secretory (E-S) proteins. However, these serological methods cannot be efficiently applied at early stage of infection. The aim of the current study was to identify the common and species-specific E-S proteins of T. spiralis and T. britovi adult worms which could have potential for accurate diagnosis of Trichinella infection at an early stage of invasion. Different sets of immunoreactive proteins were identified in T. spiralis and T. britovi proteomes by a combination of two-dimensional electrophoresis (2-DE), immunoblot and LC-MS/MS analysis. Polyubiquitin-B, a possible enoyl-CoA hydratase/isomerase YngF or polyubiquitin-like protein was found to be common for both species; gene ontology analysis confirmed its involvement in proteolysis, oxidation-reduction and translation processes, as well as in molecular transport. These molecules, being secreted or excreted at an early stage of parasite development, may play a critical role in the processes occurring during the initial steps of the host invasion and hence be suitable for diagnostic test development.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes.

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC-MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC-MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.

Acute phase protein pattern and antibody response in pigs experimentally infected with a moderate dose of Trichinella spiralis, T. britovi, and T. pseudospiralis.

The aim of the present study was to evaluate the acute-phase protein (APP) response in three groups of pigs experimentally infected with a moderate infective dose, i.e. 1000 muscle larvae (ML) of Trichinella spiralis, 3000 ML of Trichinella britovi, and 2000 ML of Trichinella pseudospiralis. Over a 62-day period of infection, we examined the serum level and kinetics of the haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA), and pig major acute-phase protein (pig-MAP). In addition, to better understand the immune response of pigs experimentally infected with three different species of Trichinella, the kinetics of IgG and IgM antibodies against excretory-secretory (ES) antigens of Trichinella ML were also investigated. In order to assess anti-Trichinella IgG dynamics, we used a commercial and an in-house ELISA based on both heterologous (T. spiralis) and homologous (T. spiralis, T. britovi, and T. pseudospiralis) Trichinella species ES antigens. Among the four APPs analyzed, the concentration of CRP and pig-MAP significantly increased only in T. britovi-infected swine when compared with control pigs. This took place as early as 6 days post-infection (dpi). Hp was the only APP whose concentration significantly increased in pigs infected with T. pseudospiralis, this occurring as late as on day 62 pi. Despite the statistical differences found, increases in pig-MAP, CRP, and Hp levels were rather mild and transitory; none of these proteins were found to be elevated in the serum of all experimental groups of pigs at the same time point after infection. Specific IgG antibodies against ES antigens of Trichinella ML were first detected by the commercial and in-house T. spiralis ML ES-antigen ELISAs on days 30, 36 and 36 pi in pigs experimentally infected with T. spiralis, T. britovi, and T. pseudospiralis, respectively. However, seroconversion in pigs experimentally infected with T. britovi was detected slightly earlier (30 dpi) when the ELISA based on homologous rather than heterologous ES antigens was applied. In serum samples from pigs infected with T. spiralis, statistically significant increases in the level of specific IgM antibodies against T. spiralis ML ES antigens were first detected on day 30 pi and after this time, their concentration began to decrease. No changes in the level of anti-Trichinella IgM were observed in T. britovi- or T. pseudospiralis-infected pigs throughout the entire period of the experiment.

Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Immunization with a Recombinant Protein of Trichinella britovi 14-3-3 Triggers an Immune Response but No Protection in Mice.

14-3-3 proteins are present in all eukaryotic organisms and are ubiquitously expressed in a broad range of tissues and cellular compartments. They are regulatory adapter proteins that play key roles in a variety of signaling pathways, and have been proposed as suitable targets for the control and detection of certain parasites. Trichinella britovi is a widely-distributed parasitic nematode, transmitted through ingestion of meat products containing invasive larvae. The present study describes the cloning and expression of Tb14-3-3, and investigates the immunological and protective potential of the recombinant protein. Immunization of mice with rTb14-3-3 triggered an IgG response, and significant differences, in the profiles of secreted cytokines observed in vitro, between experimental groups. Nonetheless, neither specific antibodies, nor increased secretion of IFNγ, IL-4, and IL-10 cytokines, conferred greater protection against infection. No reduction in larval burden was observed during recovery at 48 dpi. Additionally, rTb14-3-3 was not recognized by sera from the infected control mice, except for one, suggesting some mismatch between native and recombinant Tb14-3-3 antigenic sites. Therefore, before 14-3-3 can be considered a potential tool for Trichinella detection and vaccination, more research regarding its target proteins, and actual specific function, is needed.

Molecular identification of sarcocysts from tissue of fallow deer (Dama dama) farmed in the open pasture system based on ssu rRNA gene.

PURPOSE: Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system. METHODS: Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates. RESULTS: Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus. CONCLUSIONS: These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.

Ashworthius sidemi in cattle and wild ruminants in Poland - the current state of play.

Ashworthius sidemi, a blood-sucking abomasal nematode, has been identified in various wild ruminants, including deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and moose (Alces alces). Although it has been observed throughout Poland, most sightings have been in the eastern part of the country. However, more recently, A. sidemi has been confirmed in the Ruszów Forest District (Lower Silesian Wilderness). It is now possible to test the faeces of cattle for the DNA of the third-stage infectious larvae (L3) of A. sidemi. The present paper describes such a molecular study of 120 faecal samples collected from cattle grazed in the Ruszów Forest District and Biebrza Marshland, where A. sidemi had previously been detected in wildlife. In this study, no A. sidemi DNA was identified in any of the examined samples.

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast.

Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.