Identification of a novel methyltransferase-type 12 protein from Haemonchus contortus and its effects on functions of goat PBMCs.

BACKGROUND: Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet. RESULTS: An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-ß1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. CONCLUSIONS: Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

Toxoplasma gondii Elongation Factor 1-Alpha (TgEF-1α) Is a Novel Vaccine Candidate Antigen against Toxoplasmosis.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all warm-blood animals, leading to toxoplasmosis. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, the recombinant T. gondii elongation factor 1-alpha (rTgEF-1α) was successfully expressed in in Escherichia coli. Passive immunization of mice with anti-rTgEF-1α polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased the survival time compared with PBS control group. The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-1α PcAb also was significantly increased. Invasion of tachyzoites into mouse macrophages was significantly inhibited in the anti-rTgEF-1α PcAb pretreated group. Mice vaccinated with rTgEF-1α induced a high level of specific anti-T. gondii antibodies and production of IFN-gamma, interleukin-4. The expression levels of MHC-I and MHC-II molecules as well as the percentages of CD4+ and CD8+ T cells in mice vaccinated with rTgEF-1α was significantly increased, respectively (P < 0.05), compared with all the controls. Immunization with rTgEF-1α significantly (P < 0.05) prolonged survival time (14.53 ± 1.72 days) after challenge infection with the virulent T. gondii RH strain. These results indicate that T. gondii EF-1α plays an essential role in mediating host cell invasion by the parasite and, as such, could be a candidate vaccine antigen against toxoplasmosis.

The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina.

The gene of Eimeria acervulina microneme protein 3 (EaMIC3) was cloned and characterized. According to the conserved sequence, the 3'- and 5'-ends of EaMIC3 were amplified by the rapid amplification of cDNA ends (RACE). The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC3 was 2,607 bp and encoded a protein of 868 amino acids with 93.04 kDa. Western blotting assay showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina, whereas the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of EaMIC3. Immunofluorescence analysis indicated that EaMIC3 was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of EaMIC3 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anticoccidial index (ACI) more than 165. Moreover, EaMIC3 protein produced significantly high level of IgG antibody, IFN-γ, IL-10, IL-17 TGF-ß, CD4+ , and CD8+ .