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ABSTRACT: Chicken coccidiosis has inflicted significant economic losses upon the poultry industry. The primary strategies for preventing and controlling chicken coccidiosis include anticoccidial drugs and vaccination. However, these approaches face limitations, such as drug residues and resistance associated with anticoccidial drugs, and safety concerns related to live vaccines. Consequently, the urgent development of innovative vaccines, such as subunit vaccines, is imperative. In previous study, we screened 2 candidate antigens: Eimeria maxima lysophospholipase (EmLPL) and E. maxima regulatory T cell inducing molecule 1 (EmTregIM-1). To investigate the immune protective effect of the 2 candidate antigens against Eimeria maxima (E. maxima) infection, we constructed recombinant plasmids, namely pET-28a-EmLPL and pET-28a-EmTregIM-1, proceeded to induce the expression of recombinant proteins of EmLPL (rEmLPL) and EmTregIM-1 (rEmTregIM-1). The immunogenic properties of these proteins were confirmed through western blot analysis. Targeting EmLPL and EmTregIM-1, we developed subunit vaccines and encapsulated them in PLGA nanoparticles, resulting in nano-vaccines: PLGA-rEmLPL and PLGA-rEmTregIM-1. The efficacy of these vaccines was assessed through animal protection experiments. The results demonstrated that rEmLPL and rEmTregIM-1 were successfully recognized by anti-E. maxima chicken sera and His-conjugated mouse monoclonal antibodies. Immunization with both subunit and nano-vaccines containing EmLPL and EmTregIM-1 markedly mitigated weight loss and reduced oocyst shedding in chickens infected with E. maxima. Furthermore, the anticoccidial indexes (ACI) for both rEmLPL and PLGA-rEmLPL exceeded 160, whereas those for rEmTregIM-1 and PLGA-rEmTregIM-1 were above 120 but did not reach 160, indicating superior protective efficacy of the rEmLPL and PLGA-rEmLPL formulations. By contrast, the protection afforded by rEmTregIM-1 and PLGA-rEmTregIM-1 was comparatively lower. Thus, EmLPL is identified as a promising candidate antigen for vaccine development against E. maxima infection.

Abstract Background For decades, studies have demonstrated the anti‐inflammatory potential of proteins secreted by helminths in allergies and asthma. Previous studies have demonstrated the immunomodulatory capabilities of Succinate Coenzyme A ligase beta‐like protein (SUCLA‐β) derived from Trichinella spiralis, a crucial excretory product of this parasite. Objective To explore the therapeutic potential of SUCLA‐β in alleviating and controlling ovalbumin (OVA)‐induced allergic asthma, as well as its influence on host immune modulation. Methods In this research, we utilized the rTs‐SUCLA‐β protein derived from T. spiralis to investigate its potential in mitigating airway inflammation in a murine model of asthma induced by OVA sensitization/stimulation, both pre‐ and post‐challenge. The treatment's efficacy was assessed by quantifying the extent of inflammation in the lungs. Results Treatment with rTs‐SUCLA‐β demonstrated efficacy in ameliorating OVA‐induced airway inflammation, as evidenced by a reduction in eosinophil infiltration, levels of OVA‐specific Immunoglobulin E, interferon‐γ, interleukin (IL)‐9, and IL‐17A, along with an elevation in IL‐10. The equilibrium between Th17 and Treg cells plays a pivotal role in modulating the abundance of inflammatory cells within the organism, thereby ameliorating inflammation and alleviating symptoms associated with allergic asthma. Conclusions and Clinical Relevance Our data revealed that T. spiralis‐derived Ts‐SUCLA‐β protein may inhibit the allergic airway inflammation by regulating host immune responses.

Abstract Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

Background: Haemonchus contortus (H. contortus), a nematode with global prevalence, poses a major threat to the gastrointestinal health of sheep and goats. In an effort to combat this parasite, a nanovaccine was created using a recombinant ADP-ribosylation factor 1 (ARF1) antigen encapsulated within poly lactic-co-glycolic acid (PLGA). This study aimed to assess the effectiveness of this nanovaccine in providing protection against H. contortus infection. Methods: Fifteen goats were randomly divided into three groups. The experimental group received two doses of the PLGA encapsulated rHcARF1 (rHcARF1-PLGA) nanovaccine on days 0 and 14. Fourteen days after the second immunization, both the experimental and positive control groups were challenged with 8000 infective larvae (L3) of H. contortus, while the negative control group remained unvaccinated and unchallenged. At the end of the experiment on the 63rd day, all animals were humanly euthanized. Results: The results showed that the experimental group had significantly higher levels of sera IgG, IgA, and IgE antibodies, as well as increased concentrations of cytokines, such as IL-4, IL-9, IL-17, and TGF-β, compared to the negative control group after immunization. Following the L3 challenge, the experimental group exhibited a 47.5% reduction in mean eggs per gram of feces (EPG) and a 55.7% reduction in worm burden as compared to the positive control group. Conclusions: These findings indicate that the nanovaccine expressing rHcARF1 offers significant protective efficacy against H. contortus infection in goats. The results also suggest the need for more precise optimization of the antigen dose or a reassessment of the vaccination regimen. Additionally, the small sample size limits the statistical rigor and the broader applicability of the findings.

ABSTRACT: Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

Abstract Th9 cells play a crucial role in parasite immunity. The development of Th9 cells is facilitated by several cytokines. Key transcription factors, such as STAT6, STAT5, and PU.1, are known to enhance IL-9 expression during the Th9 immune response. NF-κB-mediated transduction pathways participate in the induction of IL-9. In a previous study, we unveiled a unique ribosomal protein derived from Haemonchus contortus excretory-secretory proteins (HcESPs) that interact with host Th9 cells. In the present study, the effects of the Haemonchus contortus ribosomal protein L6 domain DE-containing protein (HcL6) on IL-9 secretion, Th9 differentiation, and IL-9 transcription were assessed by employing ELISA, flow cytometry, and qPCR methodologies. The observations revealed the transcriptional upregulation of several key genes within the Th9 immune response pathway. Moreover, silencing STAT6, PU.1, and NF-κB was found to attenuate the Th9 immune response. In this study, we unveiled the Th9 immune response-inducing capabilities of HcL6 and elucidated some of its underlying mechanisms. These findings suggest that HcL6 is an immunostimulatory antigen capable of inducing the Th9 immune response. These insights could prove instrumental in identifying potential candidate antigens for the development of immunoprophylactic strategies against H. contortus infections.

ABSTRACT: IFN-γ plays a crucial role in resisting intracellular parasitic protozoa, such as Eimeria species. In our previous study, we identified 4 molecules derived from Eimeria maxima (E. maxima) that significantly inhibited IFN-γ production. However, the mechanism underlying this inhibitory effect remains unknown. In this study, we first investigated the effects of these 4 IFN-γ inhibitory molecules on the expression levels of chicken Toll-like receptors (chTLRs), IL-12, IL-10, TGF-β, and TNF-α in chicken macrophage HD11 and bone marrow-derived dendritic cells (BMDCs). The results demonstrated that these 4 inhibitory molecules significantly downregulated the mRNA levels of chTLR-2, chTLR-4, chTLR-21, and both mRNA and protein levels of IL-12. Subsequently, to clarify the effects of these 4 inhibitory molecules on the IL-12 secretion-related signaling pathways in chicken macrophages, qRT-PCR and Western blot were used to detect the changes of key molecules involved in the signaling pathways of IL-12 secretion (NF-κB, ERK1/2, p38, JNK, STAT3) following coincubation with these inhibitory molecules. Finally, RNAi was employed to verify the function of key molecules in the signaling pathway. The results revealed a significant upregulation in the expression of ERK1/2 phosphorylated protein induced by the 4 inhibitory molecules. Knockdown of the ERK1/2 gene significantly reduced the inhibitory effect of the 4 E. maxima inhibitory molecules on IL-12. These findings indicate that the 4 inhibitory molecules can inhibit the secretion of IL-12 by upregulating the expression of ERK1/2 phosphorylated protein, which is a key molecule in the ERK-MAPK pathway. Our study may contribute to elucidating the mechanisms underlying immune evasion during E. maxima infections, thereby providing new insights for the control of chicken coccidiosis.

ABSTRACT: It has been reported that infection of chicken coccidian could inhibit the production of Th1 cytokine IFN-γ, thereby evading clearance by the host immune system. The present study aimed to have a further investigation into the effects of Eimeria maxima IFN-γ inhibitory molecules (EmHPSP-2 and EmHPSP-3) on the immune function of chicken peripheral blood mononuclear cells (PBMC) and various T cell subsets. First, separated PBMC or sorted T cell subsets were used for incubation with recombinant proteins of EmHPSP-2 (rEmHPSP-2) and EmHPSP-3 (rEmHPSP-3). Subsequently, the effects of rEmHPSP-2 and rEmHPSP-3 on proliferative capacity, nitric oxide (NO) release and mRNA levels of cytokines of the above cells were detected. The sorting purity of CD8+, CD4+ CD25−, CD4+, and CD4+ CD25+ T cells was 93.01, 88.88, 87.04, and 81.26%, respectively. The NO release of PBMC was significantly inhibited by rEmHPSP-2 and rEmHPSP-3. The proliferation of PBMC and CD4+ T cells was significantly inhibited by rEmHPSP-2 and rEmHPSP-3, whereas CD8+, CD4+ CD25−, and CD4+ CD25+ T cells was significantly promoted by the 2 proteins. The 2 proteins significantly downregulated interferon-gamma (IFN-γ) mRNA level, upregulated the transcriptional levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) in PBMC. IFN-γ and IL-2 transcriptional levels were markedly inhibited in CD8+ T cells. IFN-γ transcriptional level was significantly inhibited, but IL-4 was promoted by rEmHPSP-2 and rEmHPSP-3 in CD4+ CD25− T cells. Meanwhile, the inhibitory effects of rEmHPSP-2 and rEmHPSP-3 on the transcriptional levels of IFN-γ and IL-2 were more obvious in CD4+ T cells containing CD25+ cells compared with the CD25+ cells depletion group. It was found that IL-10, TGF-β1, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) mRNA levels were significantly upregulated upon stimulation of chicken CD4+ CD25+ T cells by proteins. This study is not only of great significance to clarify the immune evasion mechanism of chicken coccidia, but also provides candidate antigen molecules for development of a novel vaccine against chicken coccidiosis.

Understanding the determinants of host and tissue tropisms among parasites of veterinary and medical importance has long posed a substantial challenge. Among the seven species of Eimeria known to parasitize the chicken intestine, a wide variation in tissue tropisms has been observed. Prior research suggested that microneme protein (MIC) composed of microneme adhesive repeat (MAR) domain responsible for initial host cell recognition and attachment likely dictated the tissue tropism of Eimeria parasites. This study aimed to explore the roles of MICs and their associated MARs in conferring site-specific development of E. acervuline, E. maxima, and E. mitis within the host. Immunofluorescence assays revealed that MIC3 of E. acervuline (EaMIC3), MIC3 of E. maxima (EmMIC3), MIC3 of E. mitis (EmiMIC3), MAR3 of EaMIC3 (EaMIC3-MAR3), MAR2 of EmMIC3 (EmMIC3-MAR2), and MAR4 of EmiMIC3 (EmiMIC3-MAR4), exhibited binding capabilities to the specific intestinal tract where these parasites infect. In contrast, the invasion of sporozoites into host intestinal cells could be significantly inhibited by antibodies targeting EaMIC3, EmMIC3, EmiMIC3, EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4. Substitution experiments involving MAR domains highlighted the crucial roles of EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 in governing interactions with host ligands. Furthermore, animal experiments substantiated the significant contribution of EmiMIC3, EmiMIC3-MAR4, and their polyclonal antibodies in conferring protective immunity to Eimeria-affiliated birds. In summary, EaMIC3, EmMIC3, and EmiMIC3 are the underlying factors behind the diverse tissue tropisms exhibited by E. acervuline, E. maxima, and E. mitis, and EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 are the major determinants of MIC-mediated tissue tropism of each parasite. The results illuminated the molecular basis of the modes of action of Eimeria MICs, thereby facilitating an understanding and rationalization of the marked differences in tissue tropisms among E. acervuline, E. maxima, and E. mitis.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can elicit a robust immune response during infection. Macrophage cells have been shown to play an important role in the immune response against T. gondii. In our previous study, the eukaryotic translation initiation factor 5A (eIF-5A) gene of T. gondii was found to influence the invasion and replication of tachyzoites. In this study, the recombinant protein of T. gondii eIF-5A (rTgeIF-5A) was incubated with murine macrophages, and the regulatory effect of TgeIF-5A on macrophages was characterized. Immunofluorescence assay showed that TgeIF-5A was able to bind to macrophages and partially be internalized. The Toll-like receptor 4 (TLR4) level and chemotaxis of macrophages stimulated with TgeIF-5A were reduced. However, the phagocytosis and apoptosis of macrophages were amplified by TgeIF-5A. Meanwhile, the cell viability experiment indicated that TgeIF-5A can promote the viability of macrophages, and in the secretion assays, TgeIF-5A can induce the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) from macrophages. These findings demonstrate that eIF-5A of T. gondii can modulate the immune response of murine macrophages in vitro, which may provide a reference for further research on developing T. gondii vaccines.

Avian coccidiosis arises from co-infection involving multiple Eimeria species, which could give rise to substantial economic losses in the global poultry industry. As a result, multivalent anticoccidial vaccines containing common Eimeria antigens offer considerable promise for controlling co-infection in clinical practice. In our previous study, Elongation factor 2 (EF2) was deemed as an immunogenic common antigen across various Eimeria species. This current investigation aimed to further assess the immunogenicity and protective efficacy of EF2 in recombinant subunit vaccine format against three Eimeria species. The EF2 gene cloned from Eimeria maxima (E. maxima) cDNA was designated as EF2 of E. maxima (EmEF2). The immunogenicity of the recombinant protein EmEF2 (rEmEF2) was assessed through Western blot analysis. The evaluation of the vaccine-induced immune response encompassed the determination of T lymphocyte subset proportions, cytokine mRNA transcription levels, and specific IgY concentrations in rEmEF2-vaccinated chickens using flow cytometry, quantitative real-time PCR (qPCR), and indirect enzyme-linked immunosorbent assay (ELISA). Subsequently, the protective efficacy of rEmEF2 was evaluated through vaccination and challenge experiments. The findings demonstrated that rEmEF2 was effectively recognized by the His-tag monoclonal antibody and E. maxima chicken antiserum. Vaccination with rEmEF2 increased the proportions of CD4+ and CD8+ T lymphocytes, elevated IL-4 and IFN-γ mRNA transcription levels, and enhanced IgY antibody levels compared to the control groups. Moreover, compared to the control groups, vaccination with rEmEF2 led to decreased weight loss, reduced oocyst outputs, and alleviated enteric lesions. Furthermore, in the rEmEF2-immunized groups, challenges with E. maxima and E. acervulina resulted in anticoccidial index (ACI) scores of 166.35 and 185.08, showing moderate-to-excellent protective efficacy. Nevertheless, challenges with E. tenella and mixed Eimeria resulted in ACI scores of 144.01 and 127.94, showing low protective efficacy. In conclusion, EmEF2, a common antigen across Eimeria species, demonstrated the capacity to induce a significant cellular and humoral immune response, as well as partial protection against E. maxima, E. acervulina, and E. tenella. These results highlight EmEF2 as a promising candidate antigen for the development of multivalent vaccines targeting mixed infections by Eimeria species.

Ribosomal protein L12 (RpL12) plays an important role in ovarian development and engorgement in vertebrates and invertebrates. However, the functional characteristics of RpL12 in ticks are not clear. Here, an open reading frame of the RpL12 gene was cloned from cDNA of Haemaphysalis longicornis. Th e sequence was analysed, and expression levels were determined in diff erent tissues and developmental stages using qPCR. To assess the immunization and challenge of ticks, the recombinant protein rRpL12+GST was used in immunological experiments. Th e results showed high conservation of RpL12 among species and comparisons of the amino acid sequence from H. longicornis. RpL12 was approximately 60% expressed in the ovary among the examined tissues of unfed adult female H. longicornis, and the expression level of RpL12 in unfed ticks was signifi cantly lower than that in H. longicornis at the egg and engorged stages. Western blotting showed that rabbit antiserum against H. longicornis adult ticks recognized RpL12, with an average egg weight of 49.94% and a 17.22% reduction in the engorged weight of adult ticks, but the mortality increased only 6.00%. Th ese results suggest that RpL12 could be used to generate anti-tick vaccines and provide novel information on the RpL12 gene of ticks, providing a better understanding of its mechanisms in reproduction and oogenesis.

Abstract Background Histidine acid phosphatase (HAP), a member of the histidine phosphatase superfamily, is widely found in parasites and is also a potential vaccine antigen or drug target. However, the biological function of HAP in Haemonchus contortus is still unclear. Methods We cloned the HAP gene from H. contortus (Hc-HAP) and expressed the purified recombinant Hc-HAP (rHc-HAP) protein. The transcription of the Hc-HAP gene in the eggs, infective third-stage larvae (L3s), exsheathed third-stage larvae (xL3s) and adults (females/males) was analyzed by quantitative real-time-PCR (qPCR). An immunofluorescence assay was also used to detect the localization of Hc-HAP expression in adult worms. The effect of rHc-HAP on the function of peripheral blood mononuclear cells (PBMCs) was observed by co-culture of rHc-HAP protein with goat PBMCs. Results The qPCR results revealed that the Hc-HAP gene was transcribed at a higher level in the L3 and xL3 stages that there were gender differences in transcription at the adult stage, with females exhibiting higher transcription than males. Moreover, Hc-HAP was mainly expressed in adult intestinal microvilli. Additionally, western blot results revealed that rHc-HAP could be detected in goat sera artificially infected with H. contortus. In the experiments, rHc-HAP bound to goat PBMCs and released nitric oxide. The rHc-HAP also induced the expression of interferon gamma (IFN-γ) and the phosphorylated STAT 1 transcription factor, while inhibiting interleukin-4 expression. Conclusions The results shows that rHc-HAP stimulated the IFN-γ/STAT1 signaling pathway and enabled polarization of PBMCs toward T-helper 1 immune responses. Graphical Abstract

Abstract Th9 cells have been shown to play crucial roles in anti-parasite immunity, pathogenic microbe infection, and allergy. Previous studies have demonstrated that Haemonchus contortus excretory and secretory proteins (HcESPs) induce the proliferation of Th9 cells and alter the transcriptional level of IL-9 as well as its related pathways in the Th9 immune response after infection. However, the exact molecule(s) in HcESPs inducing the Th9 immune response is not yet known. In this study, flow cytometry, co-immunoprecipitation (Co-IP) and shotgun liquid chromatography tandem-mass spectrometry (LC–MS/MS) were used, and a total of 218 proteins from HcESPs that might interact with goat Th9 cells were identified. By in vitro culture of Th9 cells with HcESPs, 40 binding proteins were identified. In vivo, 38, 47, 42 and 142 binding proteins were identified at 7, 15, 35 and 50 days post-infection (dpi), respectively. Furthermore, 2 of the 218 HcESPs, named DNA/RNA helicase domain containing protein (HcDR) and GATA transcription factor (HcGATA), were confirmed to induce the proliferation of Th9 cells and promote the expression of IL-9 when incubated with goat peripheral blood mononuclear cells (PBMCs). This study represents a proteomics-guided investigation of the interactions between Th9 cells and HcESPs. It provides a new way to explore immunostimulatory antigens among HcESPs and identifies candidates for immune-mediated prevention of H. contortus infection.

Abstract With a worldwide distribution, Eimeria spp. could result in serious economic losses to the poultry industry. Due to drug resistance and residues, there are no ideal drugs and vaccines against Eimeria spp. in food animals. In the current study, a bioinformatics approach was employed to design a multiepitope antigen, named NSLC protein, encoding antigenic epitopes of E. necatrix NA4, E. tenella SAG1, E. acervulina LDH, and E. maxima CDPK. Thereafter, the protective immunity of NSLC protein along with five adjuvants and two nanospheres in laying chickens was evaluated. Based on the humoral immunity, cellular immunity, oocyst burden, and the coefficient of growth, the optimum adjuvant was evaluated. Furthermore, the optimum immune route and dosage were also investigated according to the oocyst burden and coefficient of growth. Accompanied by promoted secretion of antibodies and enhanced CD4+ and CD8+ T lymphocyte proportions, NSLC proteins entrapped in PLGA nanospheres were more effective in stimulating protective immunity than other adjuvants or nanospheres, indicating that PLGA nanospheres were the optimum adjuvant for NSLC protein. In addition, a significantly inhibited oocyst burden and growth coefficient promotion were also observed in animals vaccinated with NSLC proteins entrapped in PLGA nanospheres, indicating that the optimum adjuvant for NSLC proteins was PLGA nanospheres. The results also suggested that the intramucosal route with PLGA nanospheres containing 300 μg of NSLC protein was the most efficient approach to induce protective immunity against the four Eimeria species. Collectively, PLGA nanospheres loaded with NSLC antigens are potential vaccine candidates against avian coccidiosis.

Haemonchus contortus is a gastrointestinal parasite that adversely impacts small ruminants, resulting in a notable reduction in animal productivity. In the current investigation, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, sourced from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The development of this nanovaccine involved the formulation of PLGA NPs using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM)verified the successful encapsulation of rHcES-15 within PLGA NPs, exhibiting a size range of 350–400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were allocated into five groups, with the nanovaccine administered on day 0 and the mice euthanized at the end of the 14-day trial. The stimulation index (SI) from the mice subjected to the nanovaccine indicated heightened lymphocyte proliferation (*** p < 0.001) and a noteworthy increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Additionally, the percentages of T-cells (CD4+, CD8+) and dendritic cell phenotypes (CD83+, CD86+) were significantly elevated (** p < 0.01, *** p < 0.001) in mice inoculated with the nanovaccine compared to control groups and the rHcES-15 group. Correspondingly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine in comparison to both the antigenic (rHcES-15) and control groups (* p < 0.05, ** p < 0.01). In conclusion, the data strongly supports the proposal that the encapsulation of rHcES-15 within PLGA NPs effectively triggers immune cells in vivo, ultimately enhancing the antigen-specific adaptive immune responses against H. contortus. This finding underscores the promising potential of the nanovaccine, justifying further investigations to definitively ascertain its efficacy.

IntroductionAvian coccidiosis, caused by apicomplexan protozoa belonging to the Eimeria genus, is considered one of the most important diseases in the intensive poultry industry worldwide. Due to the shortcomings of live anticoccidial vaccines and drugs, the development of novel anticoccidial vaccines is increasingly urgent.MethodsEimeria maxima rhomboid-like protein 1 (EmROM1), an invasion-related molecule, was selected as a candidate antigen to evaluate its protective efficacy against E. maxima in chickens. Firstly, the prokaryotic recombinant plasmid pET-32a-EmROM1 was constructed to prepare EmROM1 recombinant protein (rEmROM1), which was used as a subunit vaccine. The eukaryotic recombinant plasmid pVAX1.0-EmROM1 (pEmROM1) was constructed as a DNA vaccine. Subsequently, 2-week-old chicks were separately vaccinated with the rEmROM1 and pEmROM1 twice every 7 days. One week post the booster vaccination, induced cellular immune responses were determined by evaluating the mRNA level of cytokines including IL-2, IFN-γ, IL-4, IL-10, TGF-β, IL-17, and TNFSF15, as well as the percentages of CD4+ and CD8+ T cells from spleens of vaccinated chickens. Specific serum antibody level in the vaccinated chickens was determined to assess induced humoral immune responses. Finally, the protective efficacy of EmROM1 was evaluated by a vaccination-challenge trial.ResultsEmROM1 vaccination significantly upregulated the cytokine transcription levels and CD4+/CD8+ T cell percentages in vaccinated chickens compared with control groups, and also significantly increased the levels of serum-specific antibodies in vaccinated chickens. The animal trial showed that EmROM1 vaccination significantly reduced oocyst shedding, enteric lesions, and weight loss of infected birds compared with the controls. The anticoccidial index (ACI) from the rEmROM-vaccination group and pEmROM1-vaccination group were 174.11 and 163.37, respectively, showing moderate protection against E. maxima infection.DiscussionEmROM1 is an effective candidate antigen for developing DNA or subunit vaccines against avian coccidiosis.

Abstract Background Trehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway. The accumulation of TPP6 inside the body is harmful to the pathogen, but almost nothing is currently known about the function of TPP6 from Haemonchus contortus (CRE-GOB-1). Methods The H. contortus CRE-GOB-1 (HcGOB) gene was cloned and recombinant protein of GOB (rHcGOB) was expressed; transcription of the HcGOB gene at different developmental stages of H. contortus was then studied. The spatial expression pattern of the HcGOB gene in adult female and male worms was determined by both quantitative real-time PCR (qPCR) and immunofluorescence. The binding of the rHcGOB protein to goat PBMCs was assessed by immunofluorescence assay. The immunomodulatory impacts of rHcGOB on cell proliferation, nitric oxide generation and cytokine secretion were assessed by co-culture of rHcGOB protein with goat PBMCs. Results The HcGOB protein was transcribed in eggs, infective third-stage larvae (iL3s) and adults of H. contortus, with the highest transcript levels found in the egg stage. The transcript levels were significantly elevated in iL3s after manual desheathing. HcGOB was widely distributed in adult worms where it was mainly localized in the gut and gonads. rHcGOB was observed to bind to PBMCs and also to be recognized by sera collected from a goat infected with H. contortus. rHcGOB significantly activated the interleukin-10/transforming growth factor β/signal transducer and activator of transcription 3 (IL-10/TGF-β/STAT3) pathway in PBMCs while suppressing the transcription and expression of IL-4 and IL-17. Conclusions These results suggest that the HcGOB gene plays an important role in the development, parasitism and reproduction of H. contortus. The rHcGOB protein affected the immunomodulatory function of PBMCs in the in vitro study, suggesting that this protein would be a promising vaccine target. Graphical Abstract

The epidemic of avian coccidiosis seriously threatens the animals’ welfare and the economic gains of the poultry industry. Widespread in avian coccidiosis, Eimeria mitis (E. mitis) could obviously impair the production performance of the infected chickens. So far, few effective vaccines targeting E. mitis have been reported, and the nanovaccines composed of nanospheres captured our particular attention. At the present study, we construct two kinds of nanospheres carrying the recombinant E. mitis actin depolymerizing factor (rEmADF), then the characterization was then analyzed. After safety evaluation, the protective efficacy of rEmADF along with its nanospheres were investigated in chickens. The promoted secretions of antibodies and cytokines, as well as the enhanced percentages of CD4+ and CD8+ T cells were evaluated by the ELISA and flow cytometry assay. In addition, the absolute quantitative real-time PCR (qPCR) assay implied that vaccinations with rEmADF-entrapped nanospheres could significantly reduce the replications of E. mitis in feces. Compared with the rEmADF-loaded chitosan (EmADF-CS) nanospheres, the PLGA nanospheres carrying rEmADF (EmADF-PLGA nanosphers) were more effective in up-regulating weight efficiency of animals and generated equally ability in controlling E. mitis burdens in feces, suggesting the PLGA and CS nanospheres loaded with rEmADF were the satisfactory nanovaccines for E. mitis defense. Collectively, nanomaterials may be an effective antigen delivery system that could help recombinant E. mitis actin depolymerizing factor to enhance immunoprotections in chicken against the infections of E. mitis.

Haemonchus contortus is an important parasitic nematode of ruminants. Previous studies showed that H. contortus escape the immunity through complex mechanisms, including releasing excretory/secretory proteins (ESPs) to modulate the host immune response. However, the detailed mechanism through which H. contortus excretory/secretory proteins (HcESPs) promote immune evasion remains unknown. In the present study, we demonstrated that HcESPs inhibit the adaptive immune response of goats including downregulation of immune cell antigen presentation, upregulation of immune checkpoint molecules, activation of the STAT3/PD-L1 pathway, and activation of immunosuppressive regulatory T (Treg) cells. Furthermore, HcESPs reversed the LPS-induced upregulation of pro-inflammatory mediators in PBMCs by inhibiting the TLR4/NF-κB/MAPKs/NLRP3 signaling pathway. Our study provides a better understanding of the evasion mechanisms for H. contortus, which could be helpful in providing an alternative way to prevent the infection of this parasite.

Trichinella spiralis (T. spiralis), a nematode parasite, is the major cause of Trichinellosis, a zoonotic disease. A key role of MAPR in the reproductive system is to maintain pregnancy. Previous studies found that antihormone drug design and vaccine therapy of recombinant protein (rTs-MAPRC2) control T. spiralis infection. The current study investigates the inhibitory effects of different ratios of antibodies against Ts-MAPRC2 on the development of muscle larvae (ML) and newborn larvae (NBL). First, we performed indirect immunofluorescence assays and examined the effects of rTs-MAPRC2-Ab on ML and NBL in vitro as well as in vivo. Afterward, siRNA-Ts-MAPRC2 was transfected into T. spiralis muscle larvae. Following that, Ts-MAPRC2 protein was detected by Western Blotting, and mRNA levels were determined by qPCR. We also assessed whether siRNA-treated NBLs were infective by analyzing muscle larvae burden (MLs). Our results showed that rTs-MAPRC2-Ab greatly inhibited the activity of the Ts-MAPRC2 in ML and NBL of T. spiralis and rTs-MAPRC2-Ab reduced larval infectivity and survival in the host in a dose-dependent manner (1:50, 1:200, 1:800 dilutions). Furthermore, siRNA-Ts-MAPRC2 effectively silenced the Ts-MAPRC2 gene in muscle larvae (ML) in vitro, as well as in newborn larvae (NBL) of T. spiralis in vivo. In addition, siRNA-Ts-MAPRC2 (siRNA180, siRNA419, siRNA559) reduced host larval survival and infectivity significantly. This study, therefore, suggests that Ts-MAPRC2 might be a novel molecular target useful in the development of vaccines against T. spiralis infection.

Abstract Background The proliferative stage (tachyzoite) of Toxoplasma gondii (T. gondii) is critical for its transmission and pathogenesis, and a proto-oncogene eukaryotic translation initiation factor (eIF-5A) plays an important role in various cellular processes such as cell multiplication. Methods We performed a proteomic study to evaluate the specific roles of eIF-5A involved in invasion and replication of T. gondii, and both in vivo and in vitro trials using eIF-5A-interfered and wild tachyzoites were performed to verify the proteomic results. Results The results of our study showed that T. gondii eIF-5A affected tachyzoite growth and also participated in the synthesis of proteins through regulation of both ribosomal and splicing pathways. Inhibition of eIF-5A in T. gondii resulted in the downregulated expression of soluble adhesions, such as microneme protein 1 (MIC1) and MIC4, which in turn decreased the parasite population that adhered to the surface of host cells. The reduced attachment, combined with lower expression of some rhoptry proteins (ROPs) and dense granule antigens (GRAs) involved in different stages of T. gondii invasion such as ROP4 and GRA3, ultimately reduce the invasion efficiency. These processes regulated by eIF-5A eventually affect the replication of tachyzoites. Conclusions Our findings showed that eIF-5A influenced tachyzoite survival and was also involved in the process of parasite invasion and replication. These results will provide new clues for further development of targeted drugs to control T. gondii infection. Graphic abstract

Abstract Rhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-β and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

Abstract Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/β-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory–secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

A consensus is that the Th1 immune response plays a predominant role against avian coccidiosis. Therefore, an antigen with the ability to induce Th1 cytokine responses is an ideal candidate for the development of coccidiosis vaccines. In our previous study, EmARM-β, a Th1 cytokines-stimulating antigen, was screened from the cDNA expression library of Eimeria maxima (E. maxima). Herein, we verified its stimulative effects on Th1 cytokine productions and evaluated its protective efficacy against E. maxima infection. Recombinant EmARM-β protein was expressed, and eukaryotic expression plasmid pVAX1-EmARM-β was also constructed for the immunization of birds. An immunofluorescence assay was performed to detect the native form of EmARM-β protein in the stage of sporozoites. Expressions of specific transcription factors and cytokines in immunized chickens were measured using qPCR and ELISA to verify its stimulating function on Th1 cytokines. Specific IgG antibody levels and T lymphocyte subpopulation in the immunized chickens were detected using ELISA and indirect flow cytometry to determine induced immune responses. The results showed that EmARM-β native protein is massively expressed in the sporozoites stage of E. maxima. Effective stimulation from the EmARM-β antigen to T-bet and Th1 cytokines (IL-2 and IFN-γ) was observed in vivo. After being immunized with rEmARM-β or pVAX1-EmARM-β, significant promotion to the proportion of CD4+ and CD8+ T cells and the level of antigen-specific IgG antibodies in immunized chickens was also observed. Furthermore, vaccination with rEmARM-β antigen or pVAX1-EmARM-β resulted in alleviated weight loss and enteric lesion, reduced oocyst output, and higher anticoccidial index (ACI) in challenged birds. These results indicate that EmARM-β antigen can effectively stimulate the expression of Th1 cytokines and initiate host immune responses, providing moderate protective efficacy against E. maxima. Notably, EmARM-β protein is a promising candidate for developing a novel anticoccidial vaccine.

The infections of chicken coccidiosis impact the welfare of chickens and the economical production of poultry. Eimeria mitis is ubiquitous in chicken coccidiosis, and E. mitis infection can significantly affect the productivity of birds. Up to now, few efficient vaccines against E. mitis have been reported, whereas the recombinant subunit vaccines delivered by nanomaterials may elicit an encouraging outcome. Thus, in this study, we chose E. mitis 1a (Em1a) protein as the candidate antigen to generate Em1a preparations. The recombinant Em1a (rEm1a) protein was encapsulated with poly lactic-co-glycolic acid (PLGA) and chitosan (CS) nanospheres. The physical characterization of the rEm1a-PLGA and rEm1a-CS nanospheres was investigated, and the resulting nanospheres were proven to be nontoxic. The protective efficacy of rEm1a-PLGA and rEm1a-CS preparations was evaluated in E. mitis-challenged birds in comparison with two preparations containing rEm1a antigen emulsified in commercially available adjuvants. ELISA assay, flow cytometry analysis, and quantitative real-time PCR (qPCR) analysis indicated that vaccination with rEm1a-loaded nanospheres significantly upregulated the secretions of antibodies and cytokines and proportions of CD4+ and CD8+ T lymphocytes. Compared with the other three preparations, rEm1a-PLGA nanosphere was more effective in improving growth performance and inhibiting oocyst output in feces, indicating that the PLGA nanosphere was associated with optimal protection against E. mitis. Collectively, our results highlighted the advantages of nanovaccine in eliciting protective immunity and may provide a new perspective for developing effective vaccines against chicken coccidiosis.

Haemonchus contortus Y75B8A.8 (Hc8) derived from H. contortus excretory–secretory (ES) products was identified as a functional inhibitor of goat interleukin 2 (IL-2). It may act as a vaccine candidate for the development of therapeutic strategies against H. contortus infection. In this research, recombinant Hc8 (rHc8) and goat anti-rHc8 polyclonal antibodies were employed to evaluate the protective capacities of Hc8 antigen against H. contortus infections via active and passive immunization trials, respectively. In both trials, local crossbred female goats aged 9–12 months old were randomly divided into three groups, five in each group, respectively. Parasitological examinations, including fecal egg counts (FEC), cumulative FEC (cFEC), and worm burdens, were performed. In addition, antibody levels in mucosal homogenate (MH) samples and hematological and immunological parameters were detected. In the passive trial, goats were intravenously immunized with 5 mg total IgG containing anti-rHc8 goat polyclonal antibodies. After twice immunization, compared with the challenged control group, cFEC was reduced by 39%. In addition, there was a 46% reduction of worm burdens compared with the challenged controls. In the active immunization trials, 500 μg of recombinant Hc8 protein was given subcutaneously twice to 9–12-month-old local crossbred female goats with a 2-week interval, resulting in the generation of high levels of antigen-specific circulating antibodies. Besides, cFEC and abomasal worm burden were reduced by 70 and 55%, respectively, compared with the challenged control group. In addition, immunized goats had higher mucosal homogenate IgA and hemoglobin levels than the challenged controls in both passive and active immunization trials. These preliminary results demonstrated the immunoprophylactic effects of Hc8 antigen and will inform new studies on ES proteins in developing subunit recombinant vaccines against H. contortus.

Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimulatory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upregulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-γ secretions after incubation with recombinant proteins. While transforming growth factor-β was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eimeria infection.

Abstract 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-β. 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.

Abstract Background Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo. Methods In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion. Results We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression. Conclusions These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.

Abstract Background Gastrointestinal nematodes could release excretory-secretory (ES) proteins into the host environment to ensure their survival. These ES proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions, especially in chronic infections. In our preliminary study, Haemonchus contortus adhesion-regulating molecule 1 (HcADRM1) was identified from H. contortus ES proteins (HcESPs) that interacted with host T cells via liquid chromatography-tandem mass spectrometry analysis. However, little is known about HcADRM1 as an ES protein which may play a pivotal role at the parasite-host interface. Methods Based on bioinformatics approaches, multiple amino acid sequence alignment was conducted and the evolutionary relationship of HcADRM1 with ADRM1 orthologues was extrapolated. Employing RT-qPCR and immunohistochemistry assays, temporal transcriptional and spatial expression profiles of HcADRM1 were investigated. Using immunostaining approaches integrated with immunological bioassays, the immunomodulatory potentials of HcADRM1 on goat T cells were assessed. Results We hereby demonstrated that HcADRM1 with immunodiagnostic utility was a mammalian ADRM1 orthologue abundantly expressed at all developmental stages of H. contortus. Given the implications of ADRM1 proteins in cell growth, survival and development, we further investigated the immunomodulatory property of HcADRM1 as an individual ES protein acting at the parasite-host interface. The rHcADRM1 stimuli notably suppressed T cell viability, promoted intrinsic and extrinsic T cell apoptosis, inhibited T cell proliferation and induced cell cycle arrest at G1 phase. Simultaneously, rHcADRM1 stimuli exerted critical controls on T cell cytokine secretion profiles, predominantly by restraining the secretions of interleukin (IL)-4, IL-10 and interferon-gamma. Conclusions Importantly, HcADRM1 protein may have prophylactic potential for anti-H. contortus vaccine development. Together, these findings may contribute to the clarification of molecular and immunomodulatory traits of ES proteins, as well as improvement of our understanding of parasite immune evasion mechanism in H. contortus-host biology.

Abstract Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.

Abstract Haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. The excretory-secretory (ES) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. In this study, we identified 114 H. contortus ES proteins (HcESPs) interacting with host T cells and 15 T cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. Based on bioinformatics analysis, we demonstrated that HcESPs could inhibit T cell viability, induce cell apoptosis, suppress T cell proliferation and cause cell cycle arrest. Furthermore, the stimulation of HcESPs exerted critical control effects on T cell cytokine production profiles, predominantly promoting the secretion of interleukin (IL)-10, IL-17A and transforming growth factor-β1 and inhibiting IL-2, IL-4 and interferon-γ production. Collectively, these findings may provide insights into the interaction between ES proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development.

Abstract 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.

Abstract 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-β1 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.

Abstract 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.

Caused by Toxoplasma gondii, toxoplasmosis has aroused great threats to public health around the world. So far, no effective vaccine or drug is commercially available, and the demands for a safe and effective therapeutic strategy have become more and more urgent. In the current study, we constructed a DNA vaccine encoding T. gondii ribosomal P2 protein (TgP2) and denoted as TgP2-pVAX1 plasmid. To improve the immunoprotection, nanomaterial poly-lactic-co-glycolic acid (PLGA) and chitosan were used as the delivery vehicle to construct TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres. Before vaccinations in BALB/c mice, TgP2-pVAX1 plasmids were transiently transfected into Human Embryonic Kidney (HEK) 293-T cells, and the expression of the eukaryotic plasmids was detected by laser confocal microscopy and Western blotting. Then the immunoprotection of naked DNA plasmids and their two nano-encapsulations were evaluated in the laboratory animal model. According to the investigations of antibody, cytokine, dendritic cell (DC) maturation, molecule expression, splenocyte proliferation, and T lymphocyte proportion, TgP2-pVAX1 plasmid delivered by two types of nanospheres could elicit a mixed Th1/Th2 immune response and Th1 immunity as the dominant. In addition, TgP2-pVAX1/PLGA and TgP2-pVAX1/CS nanospheres have great advantages in enhancing immunity against a lethal dose of T. gondii RH strain challenge. All these results suggested that TgP2-pVAX1 plasmids delivered by PLGA or chitosan nanomaterial could be promising vaccines in resisting toxoplasmosis and deserve further investigations and applications.

Trehalose phosphate synthase (TPS), a key enzyme in trehalose synthesis, is not present in mammals but critical to the viability of a wide range of lower organisms. However, almost nothing is known about the function of Hc-TPS (GT1-TPS structural domain protein from Haemonchus contortus). In this study, Hc-TPS gene was cloned and the recombinant protein (rHc-TPS) was expressed and purified. The quantitative real-time PCR (qPCR) results showed that Hc-TPS was transcribed at different stages of H. contortus, with higher levels of transcription at the molting and embryo stages. Immunofluorescence analysis showed that Hc-TPS was widely distributed in adults, but the expression was mainly localized on the mucosal surface of the intestine as well as in the embryos of female worms. The impacts of rHc-TPS on peripheral blood mononuclear cell (PBMC) proliferation, nitric oxide (NO) generation, transcriptional expression of cytokines, and related pathways were examined by co-incubating rHc-TPS with goat PBMCs. The results showed that rHc-TPS significantly inhibited PBMC proliferation and NO secretion in a dose-dependent manner. We also found that rHc-TPS activated the interleukin (IL)-10/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (IL-10/STAT3/SOCS3) axis and significantly promoted SOCS3 expression, while inhibiting interferon-gamma (INF-γ), IL-4, IL-9, and IL-2 pathways. Our findings may contribute to understanding the immune evasion mechanism for the parasite during host–parasite interactions and also help to provide ideas for discovering new drug targets.

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.

Avian Eimeria species vary in their replication location, fecundity, and pathogenicity. They are required to complete the development within the limited space of host intestines, and some synergistic or antagonistic effects occur among different Eimeria species. This study evaluated the impact of Eimeria mitis on the outcome of Eimeria necatrix or Eimeria tenella challenge infection. The severity of E. mitis/E. necatrix and E. mitis/E. tenella mixed infections were quantified by growth performance evaluation, survival rate analysis, lesion scoring, blood stool scoring, and oocyst output counting. The presence of E. mitis exacerbated the outcome of co-infection with E. tenella, causing high mortality, intestinal lesion score, and oocyst production. However, E. mitis/E. tenella co-infection had little impact on the body weight gain compared to individual E. tenella infection. In addition, the presence of E. mitis appeared not to enhance the pathogenicity of E. necatrix, although it tends to inhibit the growth of challenged birds and facilitate oocyst output and mortality in an E. mitis/E. necatrix co-infection model. Collectively, the results suggested a synergistic relationship between E. mitis and E. tenella/E. necatrix when sharing the same host. The presence of E. mitis contributed to disease pathology induced by E. tenella and might also advance the impact of E. necatrix in co-infections. These observations indicate the importance of accounting for differences in the relationships among different Eimeria species when using mixed infection models.

MicroRNAs (miRNAs), which are small, noncoding RNA molecules, play an important regulatory role in gene expression at the posttranscriptional level. Relatively limited knowledge exists on miRNAs in Rhipicephalus microplus ticks in China; however, understanding the physiology of miRNA functions and expression at different developmental stages is important. In this study, three small RNA libraries were constructed for R. microplus eggs, larvae, and female adults; miRNAs were detected during these developmental stages by high-throughput sequencing, with 18,162,337, 8,090,736, and 11,807,326 clean reads, respectively. A total of 5132 known miRNAs and 31 novel miRNAs were identified. A total of 1736 differentially expressed miRNAs were significantly different at a p-value of R. microplus and their potential targets, which has significance for understanding the biology of ticks and lays the foundation for improved understanding of miRNA functioning in the regulation of R. microplus development. These results can assist future miRNA studies in other tick species that have great significance for human and animal health.

Toxoplasma gondii (T. gondii) is an intracellular parasitic protozoan that can cause serious public health problems. However, there is no effectively preventive or therapeutic strategy available for human and animals. In the present study, we developed a DNA vaccine encoding T. gondii oxidoreductase from short-chain dehydrogenase/reductase family (TgSDRO-pVAX1) and then entrapped in chitosan and poly lactic-co-glycolic acid (PLGA) to improve the efficacy. When encapsulated in chitosan (TgSDRO-pVAX1/CS nanospheres) and PLGA (TgSDRO-pVAX1/PLGA nanospheres), adequate plasmids were loaded and released stably. Before animal immunizations, the DNA vaccine was transfected into HEK 293-T cells and examined by western blotting and laser confocal microscopy. Th1/Th2 cellular and humoral immunity was induced in immunized mice, accompanied by modulated secretion of antibodies and cytokines, promoted the maturation and MHC expression of dendritic cells, and enhanced the percentages of CD4+ and CD8+ T lymphocytes. Immunization with TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres conferred significant immunity with lower parasite burden in the mice model of acute toxoplasmosis. Furthermore, our results also lent credit to the idea that TgSDRO-pVAX1/CS and TgSDRO-pVAX1/PLGA nanospheres are substitutes for each other. In general, the current study proposed that TgSDRO-pVAX1 with chitosan or PLGA as the delivery vehicle is a promising vaccine candidate against acute toxoplasmosis.

Abstract Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections.

Abstract Background Hepatocellular carcinoma-associated antigen 59 (HCA59), which is one of the most important excretory/secretory products of Haemonchus contortus (HcESPs), is known to have antigenic functions. However, its immunomodulatory effects on host cells are poorly understood. Methods Here, we cloned the HCA59 gene and expressed the recombinant protein of HCA59 (rHCA59). Binding activities of rHCA59 to goat peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) were checked by immunofluorescence assay (IFA) and the immunoregulatory effects of rHCA59 on cytokine secretions, cell migration, cell proliferation, nitric oxide production, and changes in expression of genes in related pathways were observed by co-incubation of rHCA59 with goat PBMCs and DCs. Monocyte phagocytosis and characterization of goat blood DC subsets were detected by flow cytometry. Results The IFA results revealed that rHCA59 could bind to PBMCs and DCs. Treatment of PBMCs with rHCA59 significantly increased cellular proliferation and NO production in a dose–dependent manner, while cell migration was vigorously blocked. Treatment with rHCA59 significantly suppressed monocytes phagocytosis. The quantity of surface marker CD80 on DCs increased significantly after rHCA59 treatment. In addition, the expression of genes included in the WNT pathway was related to the differentiation and maturation of DCs, and the production of IL-10 and IL-17 produced by PBMCs was altered. Conclusions Our findings illustrated that rHCA59 could enhance host immune responses by regulating the functions of goat PBMCs and DCs, which would benefit our understanding of HCA59 from parasitic nematodes contributing to the mechanism of parasitic immune evasion.

Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles carrying recombinant TA4 protein of E. tenella (rEtTA4) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 (PLGA-rEtTA4) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8+ T lymphocytes. However, no significant differences were observed in the proportion of CD4+ T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella. Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles–immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8+ T lymphocytes compared with rEtTA4 subunit vaccine–immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.

Avian coccidiosis brings tremendous economic loss to the poultry industry worldwide. The third generation vaccine, including subunit and DNA vaccines, exhibited promising developmental prospects. In a previous study, we found rhomboid-like protein 3 of Eimeria maxima (EmROM3) was involved in infections by Eimeria species. However, the protective efficacy of EmROM3 against Eimeria maxima (E. maxima) remains unknown. In this study, chickens were intramuscularly immunized with the recombinant protein EmROM3 (rEmROM3) or pVAX1-EmROM3 to determine the EmROM3-induced immune response. The induced humoral immune response was determined by measuring serum IgG antibody levels in immunized chickens. The induced cellular immune response was detected by measuring the transcription level of immune related cytokines and the proportion of T cell subsets of the immunized chickens. Finally, the protective efficacy of the EmROM3 vaccine against E. maxima was evaluated by immunization-challenge trials. Results revealed that the purified rEmROM3 reacted with chicken anti-E. maxima serum. The recombinant plasmid of pVAX1-EmROM3 was transcribed and translated in the injected muscle from the vaccinated chickens. In experimental groups, the IgG titers, proportions of CD4+ and CD8+ T cells, and transcription level of splenic cytokines were significantly increased compared with the control groups. The immunization-challenge trial revealed that immunization with rEmROM3 or pVAX1-EmROM3 led to restored weight gain, alleviated enteric lesion, decreased oocyst output as well as the higher anticoccidial index (ACI), indicating partial protection against E. maxima. These results indicate that EmROM3 is an effective candidate antigen for developing novel vaccines against infection by E. maxima.

Parasitic infections are a major public health concern affecting millions of people universally. This review elaborates on the potential impacts of plants and their bioactive components that have been widely used in the cure of several parasitic infections of poultry. The medicinal importance of natural herbs depends upon their bioactive ingredients, which are originated from crude plants, consequently leading to the specific action on the body. Due to the limited availability of effective drugs and high cost, the development of drug resistance in several harmful parasites and microbes leads to huge economic losses in the poultry industry. This will impose the development of innovative sources for drugs to overwhelm the therapeutic failure. Moreover, the environment-friendly feed additives which can be applied as a substitute to antibiotic growth promoters (AGP) for broilers were proven. The application of natural products with therapeutic characteristics is an ancient practice that is appropriately gaining more acceptance. Globally, it is assessed that some 20,000 species of higher plants are used medicinally, although traditional medicine has a scarcity of knowledge on its efficiency and wellbeing. This review explores the usage of medicinal herbs for parasitic infections, emphasizing the recent knowledge available while detecting the research gaps which may be explored to find the usage of herbal medicines for parasitic infections in poultry. In conclusion, herbal medicines are the effective source of prime components for drug detection and the formation of phytopharmaceuticals in the control of devastating parasitic infections. There is a prerequisite to applying the traditional medicine information in clinical applications via value addition.

The α/β-hydrolase domain (ABHD) proteins belonging to α/β-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.

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-β/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-β/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-β/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-β/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-β/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-β/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-β/Smad signaling key genes.

Abstract Background Avian coccidiosis is often caused by co-infection with several species of Eimeria worldwide. Developing a multivalent vaccine with an antigen common to multiple Eimeria species is a promising strategy for controlling clinical common co-infection of Eimeria. In the previous study, 14–3-3 was identified as one of the immunogenic common antigen in E. tenella, E. acervulina and E. maxima. The aim of the present study was to evaluate the immunogenicity and protective efficacy of Ea14–3-3 in the form of DNA vaccine against infection with three species of Eimeria both individually and simultaneously. Results After vaccination with pVAX-Ea14–3-3, the Ea14–3-3 gene was transcribed and expressed in the injected muscles. Vaccination with pVAX-Ea14–3-3 significantly increased the proportion of CD4+ and CD8+ T lymphocytes and produced a strong IgY response in immunized chickens. Similarly, pVAX-Ea14–3-3 stimulated the chicken’s splenocytes to produce high levels of Th1-type (IFN-γ, IL-2) and Th2-type (IL-4) cytokines. The vaccine-induced immune response was responsible to increase weight gain, decreased the oocyst output, and alleviated enteric lesions significantly in immunized chickens as compared to control group, in addition to induce moderate anti-coccidial index (ACI). Conclusion These results indicate that Ea14–3-3 is highly immunogenic and capable to induce significant immune responses. Furthermore, Ea14–3-3 antigen can provide effective protection against infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima both individually and in combination with three Eimeria species. Significant outcomes of our study provide an effective candidate antigen for developing a multivalent Eimeria vaccine against mixed infection with various Eimeria species under natural conditions.