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.

Characteristics of Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase of the parasitic nematode Haemonchus contortus and its modulatory functions on goat PBMCs in vitro.

Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase (BLAODA), as an essential excretion of Haemonchus contortus (HcESPs), was identified to have antigenic functions. T helper-9 (Th9) cells secrete interleukin (IL)-9, a signature cytokine associated with tumour immunology, allergy and autoimmunity. Nonetheless, the understanding of modulatory functions of BLAODA on Th9 and other immune cells is limited. In this study, the BLAODA gene was cloned, and the recombinant (r) protein of BLAODA (rHcBLAODA) was expressed and immunoblotting was performed. The results revealed that HcBLAODA gene was successfully cloned and rHcBLAODA protein was expressed. The localization of rHcBLAODA was confirmed on the surface of gut sections from adult H. contortus. The rHcBLAODA protein capability to react precisely with anti-H. contortus antibodies were confirmed by immunoblotting and immunofluorescence assay (IFA). Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced Th9 cells generation, IL-9 expression, nitric oxide production and cell apoptosis while suppressing the cells proliferation and cells migration depending on the concentration. Overall, these findings suggest that rHcBLAODA protein could modulate the host immune response by inducing Th9 cells to secrete IL-9 cytokine in vitro.

Nanoparticles (PLGA and Chitosan)-Entrapped ADP-Ribosylation Factor 1 of Haemonchus contortus Enhances the Immune Responses in ICR Mice.

ADP-ribosylation factor 1 (HcARF1) is one of the Haemonchus contortus (H. contortus) excretory/secretory proteins involved in modulating the immune response of goat peripheral blood mononuclear cells (PBMC). Here, we evaluated the immunogenic potential of recombinant HcARF1 (rHcARF1) against H. contortus infection in Institute of Cancer Research (ICR) mice. Briefly, rHcARF1 was entrapped in poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NP) and injected into mice as a vaccine. Fifty-six ICR mice were assigned randomly into seven groups, with eight animals in each group, and they were vaccinated subcutaneously. At the end of the experiment (14th day), the blood and the spleen were collected from euthanized mice to detect lymphocyte proliferation, cytokine analysis, and the production of antigen-specific antibodies. Scanning electron microscope was used to determine the size, morphology, and zeta potential of nanoparticles. Flow cytometry was performed, which presented the increase percentages of CD4+ T cells (CD3e+CD4+), CD8+ T cells (CD3e+CD8+) and dendritic cells (CD11c+CD83+, CD11c+CD86+) in mice vaccinated with rHcARF1+PLGA NP. Immunoassay analysis show raised humoral (Immunoglobulin (Ig)G1, IgG2a, IgM) and cell-mediated immune response (Interleukin (IL)-4, IL-12, and IL-17, and Interferon (IFN)-γ) induced by rHcARF1+PLGA NP. Experimental groups that were treated with the antigen-loaded NP yield higher lymphocyte proliferation than the control groups. Based on these results, we could propose that the rHcARF1 encapsulated in NP could stimulate a strong immune response in mice rather than administering alone against the infection of H. contortus.

Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina modulates the functions of chicken dendritic cells to boost Th1 type immune response and stimulates autologous CD4+ T cells differentiation in-vitro.

Dendritic cells (DCs) play a pivotal role to amplify antigen-specific immune responses. Antigens that sensitize T cells via antigen-presentation by DCs could enhance the capacity of host immunity to fight infections. In this study, we tested the immunogenic profiles of chicken DCs towards Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina (EaGAPDH). Immunoblot analysis showed that recombinant EaGAPDH (rEaGAPDH) protein was successfully recognized by rat sera generated against rEaGAPDH. Interaction and internalisation of rEaGAPDH by chicken splenic-derived DCs (chSPDCs) was confirmed by immunofluorescence analysis. Flow cytometry revealed that chSPDCs upregulated MHCII, CD1.1, CD11c, CD80, and CD86 cell-surface markers. Moreover, mRNA expressions of DC maturation biomarkers (CCL5, CCR7, and CD83) and TLR signalling genes (TLR15 and MyD88) were also upregulated whereas those of Wnt signalling were non-significant compared to negative controls. rEaGAPDH treatment induced IL-12 and IFN-γ secretion in chSPDCs but had no effect on IL-10 and TGF-ß. Likewise, DC-T cell co-culture promoted IFN-γ secretion and the level of IL-4 was unaffected. Proliferation of T cells and their differentiation into CD3+/CD4+ T cells were triggered in chSPDCs-T cells co-culture system. Taken together, rEaGAPDH could promote Th1 polarization by activating both host DCs and T cells and sheds new light on the role of this important molecule which might contribute to the development of new DCs-based immunotherapeutic strategies against coccidiosis.

Unveiling the Immunomodulatory Characteristics of Haemonchus contortus Ephrin Domain Containing Protein in the Parasite-Host Interactions.

Ephrin domain containing protein (EPH), a significant excreted and secreted product (ESPs) of Haemonchus contortus, has been identified to have antigenic functions. Over the past years, a new subset of CD4 + T named as T helper 9 cells that secrete interleukin-9 (IL-9) as a signature cytokine is associated with tumor immunity and allergy. Nonetheless, the understanding of immunomodulatory roles of EPH on goat Th9 and other immune cells remains limited. Herein, EPH from H. contortus (HcEPH) was cloned and expressed in pET-28a. Immunofluorescence assay (IFA) was carried-out to localize rHcEPH within H. contortus adult worms and to bind with goat peripheral blood mononuclear cells (PBMCs). Besides, the impact of rHcEPH on signature cytokine IL-9 expression in goat PBMCs was evaluated. Flow cytometry was employed to examine Th9 cells production and cell apoptosis. The results revealed success in the expression and localization of rHcEPH in surface of adult H. contortus gut sections. According to IFA analysis, the rHcEPH protein was capable to react precisely with anti-H. contortus antibodies. Further functional analysis showed that correlation between rHcEPH and host PBMCs significantly enhanced Th9 cell differentiation, IL-9 expression, cell apoptosis efficiency, and cell migration, whereas cell proliferation was suppressed significantly depending on the concentration. Our observations indicated that rHcEPH protein is linked to modulate the host immune cells and could enhance protective immunity by inducing Th9 responses.

Immunomodulatory dynamics of excretory and secretory products on Th9 immune response during Haemonchus contortus infection in goat.

CD4+ T cells play critical roles in mediating adaptive immunity to a variety of pathogens. Recently, new subset of CD4+T named as T helper 9 cells that express the prototypical interleukin-9 (IL-9) cytokine have been recognized in human and mice models during different parasitic infections. Haemonchus contortus is a gastrointestinal nematode of small ruminants which cause high mortality in young animals. During infection, Excretory and Secretary Products (ESPs) are released in the host body. No other study has reported yet on immunomodulatory dynamics of H. contortus ESPs on Th9 immune response in vitro or in vivo. In this study, immunomodulatory effects of ESPs (5, 10, 20, 40, 80; µg/mL) incubated with goat PBMCs on Th9 cells, IL-9 immune response and TGF-ß/Smad signaling regulator were evaluated in vitro. Moreover, for in vivo study, goats were infected with different doses (P-800, P-2400, and P-8000) of H. contortus infective larva (L3) and immunomodulatory effects on Th9 cells, IL-9 immune response and TGF-ß/Smad signaling regulator were evaluated at 7, 10, 14, 18, 21, 28 Days Post Infection (DPI). Flow cytometry was performed to evaluate the effects on Th9 cells and quantitative real time polymerase chain reaction was performed to evaluate the IL-9 cytokine transcription level. Additionally, fecal egg counting was also performed in parallel to confirm the infection. All goats were dewormed at 29 DPI and all experiments were also performed at 35 DPI, one week post deworming. The finding indicated that 10, 20, 40, 80 µg/mL concentration of ESPs incubated with goat PBMCs showed significant increase in the production of Th9 cells, signature cytokine IL-9 and expression of TGF-ß/Smad signaling regulator as compared to control group in vitro.All infected groups showed significant increase in production of Th9 cells and IL-9 cytokine and expression of TGF-ß/Smad key genes at 18, 21, and 28 DPI as compared to control group. Likewise, at 14 DPI, P-2400 and P-8000 groups showed significant increase in production of Th9 cells, IL-9 cytokine and expression of TGF-ß/Smad key genes. While at 10 DPI, production of Th9 cells and IL-9 was significantly increased in P-2400 & P-8000 groups, and at 7 DPI only P-8000 showed significantly increase in IL-9 production. No immunomodulatory effects were observed at 0 and 3 DPI. Additionally, significant gradually up-regulated key genes expression of TGF-ß/Smad signaling regulator in all infected groups confirmed the above results. After deworming, production of Th9 cells, associated immune response and expression of signaling regulator in each group were significantly decreased. Based on this study, it is concluded that Th9 immune response was induced during H. contortus infection in goat by up-regulation of TGF-ß/Smad signaling key genes.

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 ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro.

Dendritic cells (DCs) are key linkages between innate immunity and acquired immunity. The antigens that promote the functions of DCs might be the effective candidates of novel vaccine. In this research, the ability of ubiquitin-conjugating enzyme (UCE), a recognized common antigens among chicken Eimeria species, to stimulate DCs of chickens were evaluated. We cloned UCE gene from Eimeria maxima (EmUCE), and its protein expression was confirmed by SDS-PAGE and western-blot. Immunofluorescence assay confirmed the binding of rEmUCE on the surface of chicken splenic-derived DCs (ChSP-DCs). Flow cytometric analysis showed that rEmUCE-treated ChSP-DCs increased MHCII, CD1.1, CD11c, CD80, and CD86 phenotypes. qRT-PCR indicated that transcript levels of maturation markers CCL5, CCR7, and CD83 in ChSP-DCs were upregulated in response to rEmUCE. Following rEmUCE treatment, chSP-DCs activated TLR signaling and inhibited Wnt signaling. Moreover, rEmUCE promoted DC-mediated T-cell proliferation in DC/T-cell co-incubation. Interestingly, CD3+/CD4+ T-cells were significantly enhanced when rEmUCE-treated chSP-DCs were co-incubated with T-cells. Cytokine secretion pattern of rEmUCE-stimulated ChSP-DCs revealed that the production of IL-12 and IFN-γ was increased whereas IL-10 and TGF-ß were unchanged. Likewise, the co-incubation of ChSP-DCs with T-cells indicated increased production of IFN-γ but not IL-4. Collectively, rEmUCE could polarize DCs to immunogenic phenotype and shift the immune cells towards Th1 response. Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.

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.

Development and Potential Application of Ras Domain Containing Protein from Haemonchus contortus for Diagnosis of Goat Infection.

Haemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide. Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination. However, limitations often occur in early or mild infections. For this purpose, serological diagnosis seems to be more accurate and reliable. Ras domain-containing protein (Ras) is one of H. contortus's excretory and secretory products (ESPs) that can be isolated from different larval stages of the nematode. In this study, the recombinant H. contortus Ras domain-containing protein (rHcRas) was expressed and purified and its diagnostic potential was evaluated. Reactions between rHcRas and goat sera were tested using Western blotting (WB). The results showed that rHcRas could be recognized by sera as early as 14 days post infection (DPI), and antibodies against rHcRas in infected goats could be maintained for over 89 days. No reaction was found between rHcRas and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immunosorbent assay (ELISA) was produced based on rHcRas. The optimal coating antigen (157 ng of rHcRas/well) and serum dilutions (1:50) were determined via checkerboard titration. Indirect ELISA based on rHcRas showed 87.5% sensitivity and 90.6% specificity. The cut-off values for this experiment were determined to be 0.324 (positive) and 0.273 (negative), respectively, and the variation coefficient (CV) was less than 15%. The results of the indirect ELISA in-field examination showed that 17.6% (9/51) of the goats were infected with H. contortus, higher than the fecal examination results (15.7%, 8/51). When compared the results of the indirect ELISA and necropsy testing, 98.0% (50/51) consistency was found. These results indicated that rHcRas was a potential antigen for the diagnosis of H. contortus infection in goats.

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.

Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats.

Haemonchus contortus, a blood-sucking nematode of ruminants, causes large economic losses worldwide. Diagnosis of infection mainly depends on the evaluation of clinical signs and fecal examination. However, this has limitations for the diagnosis of early or light infections, where serological diagnosis seems to be more accurate and reliable. In this study, the recombinant H. contortus adhesion-regulating molecule protein (rHCADRM) was expressed and purified, and its diagnostic potential was evaluated. Serum samples from goats experimentally infected with H. contortus (n = 5) were collected at 0 (before infection, negative control), 7, 14, 21, 35, 49, 63, 85, and 103 days post-infection (DPI). The reactions between rHcADRM and goat serum were tested using Western blot (WB) analysis. The results show that rHcADRM can be recognized in the serum as early as 14 DPI, and the antibody against rHcADRM in infected goat could be maintained for over 89 days. No reaction was found between rHcADRM and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immune sorbent assay (ELISA) was developed based on rHcADRM. The optimal coating antigen (279 ng of rHcADRM/well) and serum dilutions (1:50) were determined by checkerboard titration. A total of 64 serum samples, including 32 from H. contortus infection goats and 32 from helminth-free goats, were used to determine the positive (0.362) and negative (0.306) cut-off values for the ELISA. The results show this serological diagnosis method is highly sensitive (90.6%) and specific (93.75%). The coefficient of variation within run and between runs was less than 11%. To apply this indirect ELISA during field examination, 51 serum samples were randomly collected from goat farms and tested using this method. The result showed that 19.6% (10/51) of goats were infected with H. contortus, which was 100% consistent with the necropsy result, higher than that of fecal examination (15.7%, 8/51). These results indicate that rHcADRM could be a potential antigen for diagnosis of H. contortus infection 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.