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Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

ABSTRACT: To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 µg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5×10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.

SUBMITTER: Xu J

PROVIDER: S-EPMC3662057 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

Xu Jinjun J Zhang Yan Y Tao Jianping J

The Korean journal of parasitology 20130425 2

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscular ...[more]

PMID: 23710081

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Project description:BackgroundPoultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters.MethodsPopulations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts).ResultsVaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites.ConclusionsThis study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.

Project description:BACKGROUND:Apicomplexan protozoans of the genus Eimeria cause coccidiosis, one of the most economically relevant parasitic diseases in chickens. The lack of a complete understanding of molecular mechanisms in the host-parasite interaction limits the development of effective control measures. In the present study, RNA sequencing (RNA-Seq) was applied to investigate the host mRNA profiles of the cecal mucosa collected at day 5 post-infection with Eimeria maxima (EM). RESULTS:Total RNA from cecal samples of the uninfected naïve control and the EM groups was used to make libraries, generating 354,924,372 and 356,229,250 usable reads, respectively, which were assembled into a total of 386,088 high-quality unigenes (transcripts) in Trinity software. RNA-Seq analysis of cecal samples in the two groups revealed 332 upregulated and 363 downregulated genes with significant differences (P ≤ 0.05), including several significant immune-related gene families, such as the major histocompatibility complex (MHC) class I alpha chain, granzyme A and immunoglobulin subtype genes among upregulated differentially expressed genes. In addition, a total of 60 clusters of differentiation (CD) molecular genes and 570 novel genes were found. The completeness of the assembled transcriptome was further assessed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Gene ontology (GO), eggNOG and CAZy for gene annotation. The broad gene categories represented by the highly differentiated host genes suggested enrichment in immune responses, and downregulation in the metabolic pathway, MARK signaling pathway, vascular smooth muscle contraction, and proteins processing in endoplasmic reticulum after EM infection. CONCLUSIONS:Eimeria maxima induced statistically significant differences in the cecal mucosal gene expression of infected chickens. These findings provide new insights into the host-parasite interaction and enhance our understanding of the molecular mechanism of avian coccidiosis.

Project description:Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX-EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX-EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX-EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX-EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX-EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX-EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis.

Project description:BackgroundCoccidiosis is a major contributor to losses in poultry production. With emerging constraints on the use of in-feed prophylactic anticoccidial drugs and the relatively high costs of effective vaccines, there are commercial incentives to breed chickens with greater resistance to this important production disease. To identify phenotypic biomarkers that are associated with the production impacts of coccidiosis, and to assess their covariance and heritability, 942 Cobb500 commercial broilers were subjected to a defined challenge with Eimeria tenella (Houghton). Three traits were measured: weight gain (WG) during the period of infection, caecal lesion score (CLS) post mortem, and the level of a serum biomarker of intestinal inflammation, i.e. circulating interleukin 10 (IL-10), measured at the height of the infection.ResultsPhenotypic analysis of the challenged chicken cohort revealed a significant positive correlation between CLS and IL-10, with significant negative correlations of both these traits with WG. Eigenanalysis of phenotypic covariances between measured traits revealed three distinct eigenvectors. Trait weightings of the first eigenvector, (EV1, eigenvalue?=?59%), were biologically interpreted as representing a response of birds that were susceptible to infection, with low WG, high CLS and high IL-10. Similarly, the second eigenvector represented infection resilience/resistance (EV2, 22%; high WG, low CLS and high IL-10), and the third eigenvector tolerance (EV3, 19%; high WG, high CLS and low IL-10), respectively. Genome-wide association studies (GWAS) identified two SNPs that were associated with WG at the suggestive level.ConclusionsEigenanalysis separated the phenotypic impact of a defined challenge with E. tenella on WG, caecal inflammation/pathology, and production of IL-10 into three major eigenvectors, indicating that the susceptibility-resistance axis is not a single continuous quantitative trait. The SNPs identified by the GWAS for body weight were located in close proximity to two genes that are involved in innate immunity (FAM96B and RRAD).

Dataset Information

Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

Publications

Efficacy of a DNA vaccine carrying Eimeria maxima Gam56 antigen gene against coccidiosis in chickens.

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