Elsevier

Veterinary Parasitology

Volume 258, 15 July 2018, Pages 114-123
Veterinary Parasitology

Research paper
The molecular characterization and protective efficacy of microneme 3 of Eimeria mitis in chickens

https://doi.org/10.1016/j.vetpar.2018.06.020Get rights and content

Highlights

  • This is the first report about microneme 3 from Eimeria mitis (EmiMIC3).

  • Characterization of MARs in EmiMIC3 suggests it might contribute greatly to invasion of host cells.

  • EmiMIC3 is expressed in sporozoites and merozoites and localized at the apex of E. mitis.

  • EmiMIC3 possesses good immunogenicity and could induce effective protection against E. mitis.

  • Our results provide available E. mitis protective antigen for multivalent anticoccidial vaccine.

Abstract

E. mitis is ubiquitous in clinical coccidiosis caused by mixed infection of Eimeria species and the infection by E. mitis usually significantly impairs productivity of the infected chickens. To date, however, few protective antigens from E. mitis have been reported. In this study, the molecular characterization and protective efficacy of microneme 3 of Eimeria mitis (EmiMIC3) were analyzed. EmiMIC3 gene was cloned from sporozoites of E. mitis and its MARs (microneme adhesive repeats domain) were predicted. Recombinant EmiMIC3 (rEmiMIC3) was expressed in E. coli and purified and then was analyzed by western blot with anti-E. mitis chicken serum. Meanwhile, native EmiMIC3 from sporozoites was analyzed by anti-rEmiMIC3 rat serum. The expressions of EmiMIC3 in E. mitis sporozoites and merozoites were analyzed by immunofluorescence assay. The rEmiMIC3-induced changes of T lymphocytes subpopulation, serum cytokines and IgY levels and the protective efficacy of rEmiMIC3 were determined in animal experiments. The results showed that the deduced open reading frame (ORF) of EmiMIC3 was composed of 1145 amino acids, possessing 9 MARs. EmiMIC3 gene was submitted to GenBank (accession number: MG888670). EmiMIC3 could express in sporozoites and merozoites respectively and located at the apex of E. mitis sporozoite. Western blot assay revealed that the rEmiMIC3 could be recognized by serum of chicken infected by E. mitis and the native EmiMIC3 from sporozoites could also be recognized by rat serum against rEmiMIC3. Following vaccination with rEmiMIC3, higher levels of IL-10, IFN-γ, TGF-βand IL-17, higher proportions of CD4+/CD3+ and CD8+/CD3 + T lymphocytes and higher level of IgY antibody were induced compared to the controls. Vaccination with rEmiMIC3 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection. Our result not only enriches protective candidate antigen of E. mitis, but also provides available protective antigen of E. mitis for the development of multivalent vaccines against infection caused by mixture of Eimeria species in clinical coccidiosis.

Section snippets

Introdction

Coccidiosis of domestic chickens, one of the major diseases in poultry farming, is caused by several species of the genus Eimeria and causes annual economic losses of more than $3 billion worldwide (Blake and Tomley, 2014; Witcombe and Smith, 2014). Infection of Eimeria parasite causes death and low productivity including inefficient feed utilization, poor weight gain and reduced egg production of the infected chickens. Subclinical infection of less-pathogenic Eimeria species, such as E. mitis

Animals and parasites

New-born Hy-Line layer chickens were raised under coccidia-free conditions for recovery and propagation of E. mitis parasite and experimental vaccination study. The chickens were provided with feed and water without anticoccidial drugs. SD rats of 30 days old were bought from the Comparative Medicine Centre, Yangzhou University, Yangzhou, China, for antiserum collection. All animal studies and protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural

Cloning and sequence analysis of EmiMIC3

EmiMIC3 gene was amplified from the purified E. mitis sporozoite and submitted to GenBank with an accession number of MG888670. The open reading frame (ORF) is predicted to encode an 1145-amino acid protein with a molecular weight of 124.9 kDa and a pI (protein isoelectric point) of 4.71.

Characteristic analysis of MARs in EmiMIC3

BlastP revealed that EmiMIC3 contains 9 MARs, namely MAR1 (residues 49–142), MAR2 (residues 157–250), MAR3 (residues 315–408), MAR4 (residues 456 to 547), MAR5 (residues 569–662), MAR6 (residues 677–768), MAR7

Discussion

Avian coccidiosis is caused by individual or multiple Eimeria species and causes huge economic losses to poultry industry (Blake and Tomley, 2014; Witcombe and Smith, 2014). This disease is currently controlled through a combination of good husbandry, chemoprophylaxis and/or live parasite vaccination. However, next-generation vaccines including subunit or recombinant vaccines are required (Blake et al., 2017). To date, the reported candidate antigens for developing next-generation anticoccidial

Conclusion

E. mitis is one of the most prevalent Eimeria species causing mixed infection in backyard flocks and commercial flocks. MICs play important roles in parasite adhesion and invasion of host cells. To date, the reported protective microneme protein from E. mitis is not seen. In this study, an invasion related protein of EmiMIC3 was cloned and expressed. Its molecular characterization and protective efficacy were analyzed. EmiMIC3 possesses 9 microneme adhesive repeat regions. It is expressed in

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by the Joint Research Project between National Natural Science Foundation of China and Pakistan Science Foundation (NSFC-PSF) (Grant No. 31661143017), the National Natural Science Foundation of China (Grant No. 31372428, 31672545) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

References (64)

  • B. Cowper et al.

    The molecular basis for the distinct host and tissue tropisms of coccidian parasites

    Mol. Biochem Parasitol.

    (2012)
  • R.A. Dalloul et al.

    In ovo administration of CpG oligodeoxynucleotides and the recombinant microneme protein MIC2 protects against Eimeria infections

    Vaccine

    (2005)
  • X. Ding et al.

    In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis

    Vaccine

    (2005)
  • N. Friedrich et al.

    Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites

    J Biol Chem.

    (2010)
  • R.M. Godwin et al.

    A molecular survey of Eimeria in chickens across Australia

    Vet. Parasitol.

    (2015)
  • A. Haug et al.

    A simplified protocol for molecular identification of Eimeria species in field samples

    Vet. Parasitol.

    (2007)
  • T.D. Hoan et al.

    Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti

    Exp. Parasitol.

    (2016)
  • P.A. Holdsworth et al.

    World association for the advancement of veterinary parasitology (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys

    Vet. Parasitol.

    (2004)
  • J. Huang et al.

    Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge

    Parasitol. Int.

    (2015)
  • M.C. Jenkins

    Progress on developing a recombinant coccidiosis vaccine

    Int. J. Parasitol.

    (1998)
  • K. Kundu et al.

    Humoral and cytokine response elicited during immunisation with recombinant immune mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella

    Vet. Parasitol.

    (2017)
  • M. Labbé et al.

    Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection

    Mol. Biochem Parasitol.

    (2005)
  • L.H. Lan et al.

    Prevalence and drug resistance of avian Eimeria species in broiler chicken farms of Zhejiang province

    China Poult. Sci.

    (2017)
  • L. Liu et al.

    Immunoproteomic analysis of the second-generation merozoite proteins of Eimeria tenella

    Vet. Parasitol.

    (2009)
  • K. Sasai et al.

    Dynamics of lymphocyte subpopulation changes in the cecal tonsils of chickens infected with Salmonella enteritidis

    Vet. Microbiol.

    (2000)
  • M.W. Shirley et al.

    Challenges in the successful control of the avian coccidia

    Vaccine

    (2007)
  • X. Song et al.

    Partial protection against four species of chicken coccidia induced by multivalent subunit vaccine

    Vet. Parasitol.

    (2015)
  • X. Song et al.

    Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens

    Exp. Parasitol.

    (2015)
  • X. Song et al.

    Immune protection duration and efficacy stability of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 against coccidiosis

    Res. Vet. Sci.

    (2017)
  • F. Tomley

    Techniques for isolation and characterization of apical organelles from Eimeria tenella sporozoites

    Methods

    (1997)
  • M. Wallach

    Role of antibodies in immunity and control of chicken coccidiosis

    Trends Parasitol.

    (2010)
  • C.H. Yun et al.

    Intestinal immune responses to coccidiosis

    Dev. Comp. Immunol.

    (2000)
  • Cited by (0)

    View full text