Prevalence of gastrointestinal parasitism in pigs of Tripura, India.

Pigs breeds are an important livestock species mostly reared by economically lower incomesection of people in India. Within North-Eastern (NE) states, pig husbandry is very much popular hence maintain the livelihood of the rural native population. Gastrointentinal (GI) parasitic infectionisone of the major constraint in profitable pig production in this area. In the present study, the GI parasitism was investigated in 388 pigs in the three districts of Tripura, NE State of India. The examination of faecal samples revealed 61.65% overall prevalence of parasitic infestation, precisely6 GI parasitic species; including 4 nematodes and 2 protozoa, while 46.91% were the mixed infections.Metastrongylus spp. (17.53%), Strongyloids spp. (19.33%),Trichuris spp. (15.98%), Coccidia spp. (12.37%), and Balantidium coli (10.82%), were detected, however, Ascaris spp. was the most prevalentrecording 32.47%. The epidemiological factors including: age, sex, season, breed, area and farming system wise when considered as markers of study showed the highest prevalence of GI parasites in grower(6-12 months) stage, female, monsoon season, non-descript breeds, Khowai district and free range farming system, recorded 71.52%, 67.27%, 65.78%, 65.71%, 64.57%, and 69.44%, respectively. Overall, our study provides a baseline data for further investigation and formulation of strategies for control of GI parasitism in pigs in Tripura.

Characterization of interferon gamma gene in relation to immunological responses in Haemonchus contortus resistant and susceptible Garole sheep.

Garole sheep exhibits within-breed difference in resistance to natural gastrointestinal nematode infection predominated by Haemonchus contortus. In the present study, interferon gamma gene (IFN-γ) was characterized in relation to parasitological, haematological, and immune response against H. contortus in resistant and susceptible Garole sheep. Resistant and susceptible Garole sheep were selected from the field based on consistent low faecal egg counts (FEC) for one year and single nucleotide polymorphisms (SNPs) in the IFN-γ gene. The partial amplification of IFN-γ gene (1282 bp) revealed 4 SNPs exclusively in resistant sheep and 3 SNPs were shared between resistant and susceptible Garole sheep. The selected resistant and susceptible Garole sheep were challenged with H. contortus infection. The parasitological, haematological, immunological responses, and expression of IFN-γ gene were compared between the resistant and susceptible Garole sheep. The FEC of resistant sheep was significantly (P < 0.05) lower than the susceptible sheep infected with H. contortus. There was spontaneous elimination of H. contortus from 28 to 33 days post infection (DPI) in resistant sheep. Haemoglobin and packed cell volume were significantly (P < 0.05) higher in resistant sheep than the susceptible sheep. The serum concentration of immunoglobulin (Ig)G1 and IgA and cytokine IFN-γ activity and also the expression of IFN-γ gene were significantly (P < 0.05) higher in the infected resistant sheep from 14 to 28 DPI compared to the susceptible sheep. In resistant sheep, IgA and IgG1 and cytokine IFN-γ positively correlated with expression of IFN-γ gene, and the SNPs recorded in the resistant sheep only might play an important role in conferring resistance against H. contortus. Further studies are required to elucidate the role of IFN-γ gene in H. contortus resistance in Garole sheep.

Parasitological and immunological response to Haemonchus contortus infection: Comparison between resistant Garole and susceptible Sahabadi sheep.

Parasitological and immunological responses to the experimentally induced Haemonchus contortus infection were compared between Garole and Sahabadi breeds of sheep. The experiment was conducted in a 2 (breed) × 2 (infection status) factorial arrangement with a completely randomised design. Two breeds of sheep were divided into infected (n = 10) and control (n = 6) groups, and the infected groups were orally infected with H. contortus (500 stage 3 larvae per kilogram of body weight). Faecal egg counts (FEC) were determined from 18 days post infection (DPI) at 3-day intervals until 42 DPI. Average daily body weight gain, packed cell volume (PCV), concentrations of serum immunoglobulin (Ig) G1, IgG2, IgE and peripheral eosinophil count were measured at 14-day intervals from 0 to 42 DPI. Lymphocyte proliferation in response to somatic antigen of H. contortus was determined by in vitro lymphoproliferation assay, and concentrations of interferon gama (IFN-γ) and interleukin 4 (IL-4) in lymphocyte culture supernatant were measured at 14-day intervals until 42 DPI. Variables were analysed using the repeated measures mixed model procedure over DPI. Faecal egg count was significantly (p < 0.01) lower in Garole sheep than Sahabadi sheep and no faecal eggs were detected in the infected Garole sheep on 30 DPI. Infected Garole sheep had significantly (p < 0.05) higher body weight gain and PCV% than the infected Sahabadi sheep. In the infected Garole sheep, serum Ig except IgE increased significantly (p < 0.05) compared to infected Sahabadi sheep. On 28 DPI, peripheral eosinophil number, in vitro lymphoproliferation as well as concentrations of IFN-γ and IL-4 in culture supernatant were significantly (p < 0.05) higher in the infected Garole sheep than in the infected Sahabadi sheep. Parasitological observations indicated that Garole sheep were resistant to H. contortus and they exhibited greater cellular as well as humoral immune responses compared to Sahabadi sheep.

Protective antibody and cytokine responses in mice following immunization with recombinant beta-tubulin and subsequent Trypanosoma evansi challenge.

BACKGROUND: Trypanosomosis or Surra, caused by the flagellated hemoprotozoan parasite Trypanosoma evansi, is a disease of economic importance through its wide prevalence in domestic livestock in tropical countries. In the absence of a protective vaccine, management of the disease relies on a few available chemotherapeutic agents. Although humoral immunity is the mainstay of resistance to T. evansi, the ability of the parasite to vary its immunodominant surface proteins to subvert the immune system has forced vaccine efforts to target a variety of invariant epitopes. Beta tubulin, an integral component of the trypanosome cytoskeleton, was therefore targeted using the recombinant form of the protein for immunization. METHODS: The 1329 bp coding sequence of beta tubulin gene was PCR amplified and cloned in pQE-TriSystem expression vector. Recombinant beta tubulin was heterologously expressed in Escherichia coli as a 46 KDa fusion protein and used for immunization of mice. The Ig response was studied by ELISA, whereas the cytokine response was measured using a cytometric bead-based assay quantified by flow cytometry. RESULT: Immunization with recombinant beta (ß)-tubulin protein induced a beta-tubulin specific humoral immune response of predominantly IgG2a isotype. Lethal challenge with T. evansi blood-form trypomastigotes post-immunization elicited a robust anamnestic response. An abundance of IFN-γ further confirmed the Th-1 bias of the protective response. We also observed extended survival and better control of the challenge infection in the immunized mice. CONCLUSIONS: A robust anamnestic response following challenge including a Th-1 serum cytokine profile coupled with increased survival is indicative of protective immunity in the immunized mice. These observations suggest that ß-tubulin of T. evansi is a viable antigenic target for development of a vaccine against this important livestock pathogen.

Sero-surveillance for surra in cattle using native surface glycoprotein antigen from Trypanosoma evansi.

Surra, caused by Trypanosoma evansi affects a wide range of domestic and wild animals in the tropics, taking a huge toll on the already impoverished economy here. In bovines surra normally develops into a chronic infection that is often associated with severe production losses, yet with no distinct clinical signs making its adequate diagnosis vital. Though direct microscopic observation of T. evansi in circulation may be the diagnostic gold standard for surra, it is insensitive and impractical for population prevalence studies, making sero-diagnosis the preferred choice for the latter. In this study, we standardize an ELISA with Concanavalin-A (Con-A) affinity purified T. evansi surface glycoprotein antigen and compare its sensitivity and specificity to direct microscopy of stained thin smears and molecular (PCR) diagnostics. The ELISA was then put on field trial for sero-surveillance of cattle for surra in three geographically distinct populations in the Indian subcontinent, to yield an overall sensitivity and specificity of 100% and 89.15% compared to standard stained thin smear examinations and 95.23% and 90.84% compared to blood PCR examinations.

High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis.

Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.