Anti-coccidial properties and mechanisms of an edible herb, Bidens pilosa, and its active compounds for coccidiosis.
ABSTRACT: Avian coccidiosis is an economically important disease in the poultry industry. In view of the disadvantages of anti-coccidial drugs in chickens, edible plants and their compounds are re-emerging as an alternative strategy to combat this disease. A previous publication reported that the edible plant B. pilosa showed promise for use against coccidiosis. Here, we first investigated into the anti-coccidial effects of B. pilosa. We found that B. pilosa at 100 ppm or more significantly suppressed E. tenella as evidenced by reduction in mortality rate, oocyst excretion and gut pathological severity in chickens and its minimum prophylactic duration was 3 days. Next, we explored the mode of action of anti-coccidial mechanism of B. pilosa. The E. tenella oocysts were not directly killed by B. pilosa; however, administration of the plant suppressed oocyst sporulation, sporozoite invasion, and schizonts in the life cycle of E. tenella. Besides, B. pilosa boosted T cell-mediated immunity. Finally, we characterized the related anti-coccidial phytochemicals and their mode of action. One of three potent polyynes present in B. pilsoa, Compound 1 (cytopiloyne), acted against coccidiosis in chickens in a similar manner to B. pilosa. These data illustrate the anti-coccidial potency and mechanism of B. pilosa and one of its active compounds, and provide a cornerstone for development of novel herbal remedies for avian coccidiosis.
Project description:Coccidiosis is an intestinal disorder of poultry and often caused by simultaneous infections of several Eimeria species. GAPDH is one of the immunogenic common antigens among Eimeria tenella, E. acervulina, and E. maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy. The genes of GAPDH cloned from E. acervulina and E. maxima were named as EaGAPDH and EmGAPDH, respectively. The immunogenicity of recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot. The transcription and expression of pVAX-EaGAPDH and pVAX-EmGAPDH in the injected muscles were detected by reverse transcription PCR (RT-PCR) and Western blot, respectively. GAPDH-induced changes of T lymphocytes subpopulation, cytokines production, and antibody were determined using flow cytometry, quantitative real-time PCR (qPCR), and ELISA, respectively. Finally, the protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by vaccination and challenge experiments. The results revealed that the recombinant GAPDH proteins reacted with the corresponding chicken antisera. The EaGAPDH genes were successfully transcribed and expressed in the injected muscles. Vaccination with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4+ and CD8+ T lymphocytes, the cytokines productions of IFN-?, IL-2, IL-4 et al., and IgG antibody levels compared to controls. The vaccination increased the weight gains, decreased the oocyst outputs, alleviate the enteric lesions compared to controls, and induced moderate anti-coccidial index (ACI). In conclusion, the coccidial common antigen of GAPDH induced significant humoral and cellular immune response and effective protection against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species.
Project description: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-?, 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.
Project description:The protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite's sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission.Comparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered.The need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.
Project description: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<sup>+</sup> T lymphocytes. However, no significant differences were observed in the proportion of CD4<sup>+</sup> 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<sup>+</sup> 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.
Project description:Coccidiosis is one of the most economically important diseases affecting the poultry industry. Currently, anticoccidial drugs used in veterinary clinics show many deficiencies, and new control measures are urgently needed. This study presents an anticoccidial herbal powder "Shi Yin Zi", which consists of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand.-Mazz., and sodium chloride. In chickens infected with Eimeria tenella, supplementation with "Shi Yin Zi" powder for 3 d prior to infection or treatment with "Shi Yin Zi" powder after infection could improve the survival rate and relative growth rate and alleviate the pathological changes in the cecum, liver, and kidney. "Shi Yin Zi" powder could recover the levels of alanine aminotransferase, creatinine, albumin, and triglycerides in serum. The hemorrhage occurrence and total number of oocysts in feces were reduced. The anti-coccidial indexes reached 165 for the prophylactic effect and 144 for the therapeutic effect. The anti-coccidial effects were equal to positive controls (monensin and sulfamlopyrazine). These results suggest that "Shi Ying Zi" powder possesses a potent anticoccidial effect and exhibits the potential to control E. tenella infection.
Project description:Coccidiosis, caused by parasites of the genus Eimeria, is one of the most widespread and economically detrimental diseases in the global poultry industry. Because the merozoite stage of Eimeria tenella is immunologically vulnerable, motile, and functionally important for the parasites, the proteins expressed in these stages are considered to be potentially immunoprotective antigens, especially the secreted antigens and surface antigens. Here, we detected a previously unidentified MIC2-associated protein (Et-M2AP) from E. tenella and determined its localization. An immunofluorescence assay revealed that Et-M2AP was distributed in the apical part of second generation merozoites and sporozoites. In addition, an expression profile analysis revealed that the transcriptional level of Et-M2AP is significantly higher in the merozoite stage. To assess the potential of Et-M2AP protein as a coccidiosis vaccine, we expressed recombinant Et-M2AP (rEt-M2AP) and compared the immune protective efficacy of rEt-M2AP with 3 surface antigens that are highly expressed by merozoites (rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins). The immune protective efficacy of these vaccine candidates was assessed based on survival rate, lesion score, BW gain, relative BW gain, and oocyst output. The results show that the survival rate was 90%, which are significantly higher than those in the challenge control group. The BW gain rate was 42% (P < 0.001) in rEt-M2AP-immunized chickens, which are significantly higher than those in the challenge control group and rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins-immunized chickens. In addition, chickens immunized with rEt-M2AP (88% oocyst output decrease rate, P < 0.001) had the least oocyst output, compared with those immunized with rEt-SAG16 (59.2% oocyst output decrease rate, P < 0.001), rEt-SAG23 (22% oocyst output decrease rate), and rEt-SAG2 (1.36% oocyst output decrease rate). These results demonstrate that rEt-M2AP provided effective protection against challenge with E. tenella, suggesting that rEt-M2AP is a promising candidate antigen gene for development as a coccidiosis vaccine.
Project description:Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.
Project description:Avian coccidiosis is a disease caused worldwide by several species of parasite Eimeria that causes significant economic losses. This disease affects chickens development and production, that most of times is controlled with anticoccidial drugs. Although efforts have been made to address this disease, they have been made to control Eimeria sporozoites, although enteric stages are often vulnerable, however; the parasite oocyst remains a problem that must be controlled, as it has a resistant structure that facilitates dispersion. Despite some commercial products based on chemical compounds have been developed as disinfectants that destroy oocysts, the solution of the problem remains to be solved.In this work, we assessed in vitro anticoccidial activity of a compound(s) secreted by yeast isolated in oocysts suspension from infected chickens. The yeast was molecularly identified as Meyerozyma guilliermondii, and its anticoccidial activity against Eimeria tenella oocysts was assessed. Here, we report the damage to oocysts walls caused by M. guilliermondii culture, supernatant, supernatant extract and intracellular proteins. In all cases, a significant decreased of oocysts was observed.The yeast Meyerozyma guilliermondii secretes a compound with anticoccidial activity and also has a compound of protein nature that damages the resistant structure of oocyst, showing the potential of this yeast and its products as a feasible method of coccidiosis control.
Project description:Chicken coccidiosis is a protozoan parasitic disease that leads to considerable economic losses in the poultry industry. In this study, we used invasive Lactobacillus plantarum (L.P) expressing the FnBPA protein as a novel bacterial carrier for DNA delivery into epithelial cells to develop a live oral DNA vaccine. A fusion DNA vaccine co-expressing EtMIC2 and chicken IL-18 (chIL-18) was constructed and then delivered to the host by invasive L.P. Its efficacy against Eimeria tenella challenge was evaluated in chickens by examining the relative weight gain rate; caecal lesion score; OPG; anti-coccidial index (ACI); levels of EtMIC2 antibody, FnBPA, IL-4, IL-18, IFN-? and SIgA; and proliferation ability and percentages of CD4+ and CD8+ splenocytes. The experimental results showed that chickens immunized with invasive L.P carrying the eukaryotic expression vector pValac-EtMIC2 (pValac-EtMIC2/pSIP409-FnBPA) had markedly improved immune protection against challenge compared with that of chickens immunized with non-invasive L.P (pValac-EtMIC2/pSIP409). However, invasive L.P co-expressing EtMIC2 with the chIL-18 vector exhibited the highest protection efficiency against E. tenella. These results indicate that invasive Lactobacillus-expressing FnBPA improved humoural and cellular immunity and enhanced resistance to E. tenella. The DNA vaccine delivered by invasive Lactobacillus provides a new concept and method for the prevention of E. tenella.
Project description:BACKGROUND: Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis. METHODS: Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively. RESULTS: A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts. CONCLUSIONS: The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.