Trichinella spiralis: Knockdown of gamma interferon inducible lysosomal thiol reductase (GILT) results in the reduction of worm burden.

Trichinella spiralis is mammalian skeletal muscles parasite which may cause trichinellosis in animals and humans. Gamma interferon inducible lysosomal thiol reductase (GILT) is a widespread superfamily which plays key role in processing and presentation of MHC class II restricted antigen by catalyzing disulfide bond reduction. There are no reports about GILT in T. spiralis. In present study, GILT from T. spiralis (Tsp-GILT) was cloned, analyzed by multiple-sequence alignment, and predicted by 3D structure model. Recombinant Tsp-GILT (about 46 kDa) was efficiently expressed in Escherichia coli and thiol reductase activity suggested that in acidic environment the addition of a reducing agent is needed. Soaking method was used to knockdown expression of Tsp-GILT using small interference RNA (siRNA). Immunofluorescence assay confirmed the transformation of siRNA into muscle larva (ML) and new born larva (NBL). Quantitative real time-PCR (QRT-PCR) analysis revealed that transcription level of Tsp-GILT mRNA can be up-regulated by stimulation of mouse IFN-γ and down-regulated by siRNA2 in vitro. NBLs soaked with siRNA2 showed 32.3% reduction in the generation of MLs. MLs soaked with siRNA2 showed 26.2% reduction in the next generation of MLs, but no significant effect was observed on adult worms or NBLs. These findings concluded that GILT may play important roles in the development of T. spiralis parasite.

Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity.

BACKGROUND: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized. METHODS: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat. RESULTS: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. CONCLUSIONS: Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat.

BACKGROUND: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. RESULTS: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 µg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. CONCLUSION: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Galectin Domain Containing Protein from Haemonchus contortus Modulates the Immune Functions of Goat PBMCs and Regulates CD4+ T-Helper Cells In Vitro.

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN- and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.

Immunodiagnostic potential of recombinant tropomyosin during prepatent Haemonchus contortus infection in goat.

Excretory and secretory products (ESPs) are released by the parasites during Haemonchus contortus (H. contortus) infection. In this study, Tropomyosin (TpMy), one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay (ELISA) for detection of H. contortus during early infection in goat. Microscopic examination was performed parallel for comparison. Recombinant tropomyosin protein was purified successfully. Western blotting results revealed that anti-recombinant H. contortus Tropomyosin (rHc-TpMy) antibodies could recognize the natural proteinand rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica, Trichinella spiralis, and Toxoplasma gondii. Moreover, initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection (DPI) and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage (7 and 14 DPI). The optimized antigen coating concentration was calculated as 10 µg/ml (P/N Optimum Density450 = 4.165) with optimized dilution of serum (1:50) and secondary antibody (1:2500). Positive and negative cutoff value of the indirect-ELISA assay was calculated as 0.392 and 0.344, respectively. Receiver operating characteristic curve analysis validated the cutoff value (0.392) based on a high specificity and sensitivity. Indirect ELISA showed 90% diagnostic sensitivity and 100% diagnostic specificity. In comparison of serological and conventional method, rHc-TpMy based indirect ELISA showed more positive results (30%; 9/30) than microscopic examination (20%; 6/30). These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.

Succinate Coenzyme A Ligase Beta-Like Protein from Trichinella spiralis Suppresses the Immune Functions of Rat PBMCs in Vitro and Inhibits the Secretions of Interleukin-17 in Vivo.

: Succinate Coenzyme A ligase beta-like protein (SUCLA-ß) is a subunit of Succinyl-coenzyme A synthetase, which is involved in substrate synergism, unusual kinetic reaction in which the presence of SUCLA-ß for one partial reaction stimulates another partial reaction. Trichinella spiralis is a parasitic nematode, which may hinder the development of autoimmune diseases. Immunomodulatory effects of SUCLA-ß from Trichinella spiralis in the parasite-host interaction are unidentified. In this study the gene encoding T. spiralis SUCLA-ß was cloned and expressed. Binding activities of recombinant T. spiralis SUCLA-ß (rTs-SUCLA-ß) to rat peripheral blood mononuclear cells (PBMCs) were checked by immunofluorescence assay (IFA) and the immuno-regulatory effects of rTs-SUCLA-ß on cell migration, cell proliferation, nitric oxide (NO) production and apoptosis were observed by co-incubation of rTs-SUCLA-ß with rat PBMCs in vitro, while cytokine secretions in rTs-SUCLA-ß treated rats were evaluated in vivo. Furthermore, phagocytosis of monocytes was detected by flow cytometry and effects of rTs-SUCLA-ß-induced protective immunity on T. spiralis adult worms and muscle larva were evaluated in rats. The IFA results revealed that rTs-SUCLA-ß could bind to rat PBMCs. Treatment of PBMCs with rTs-SUCLA-ß significantly decreased the monocyte phagocytosis, cell migration and cell proliferation, while NO production and apoptosis of PBMCs were unaffected. Results of the in vivo study showed that the IL-17 secretion decreased significantly after rTs-SUCLA-ß administration in rats, while no significant effects were observed on the secretions of IFN-γ, IL-9, TGF-ß and IL-4. Moreover, significant reduction of T. spiralis muscle larvae burden and significant increase in anti-rTs-SUCLA-ß immunoglobulin level of IgG, IgG1 and IgG2a was observed in rTs-SUCLA-ß-administered rats. The results indicated that rTs-SUCLA-ß may be a potential target for controlling T. spiralis infection by suppressing the immune functions of the rat PBMCs and by reducing the parasite burden. Additionally it may also contribute to the treatment of autoimmune diseases and graft rejection by suppressing IL-17 immune response in the host.

Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat.

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

Prevalence and associated risk factors of haemoparasites, and their effects on hematological profile in domesticated chickens in District Layyah, Punjab, Pakistan.

Several haemoparasites commonly infect avian species, including chicken that significantly decline productivity and ultimately lead to high mortality rate. The current study was designed to assess the prevalence and associated risk factors of haemoparasites in domesticated chickens in and around District Layyah, Punjab Province, Pakistan. For this purpose, blood samples from a total of 384 backyard chickens were collected from wing vein using sterile insulin syringe. The parasites were identified from Giemsa stained thin blood smears based on morphological features using standard keys. Results demonstrated that a total of 265 backyard chickens (69%) were infected with haemoparasites in District Layyah. According to genus-wise distribution, 31.5%, 24.4% and 13% prevalence of genera Plasmodium/Haemoproteus, Leucocytozoon and mixed species were recorded, respectively. Among associated risk factors, the prevalence was relatively higher in females, chicks, naked neck breeds, scavengers feeding patterns and chickens reared at fully open coops type. The information given in the study could be of much importance in planning of an effective haemoparasites control program at District and Provincial level.