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Title:   Author  item
                                            Jenkins, Markitem
                                            Fetterer, Raymonditem
                                            Miska, Kateitem
                                            Tuo, Wenbinitem
                                            Kwok, Oliveritem
                                            Dubey, Jitender
  Submitted to: Veterinary Parasitology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 5/1/2015
                                    Publication  Date: 5/14/2015
                                    Citation: Jenkins, M.C., Fetterer, R.H., Miska, K.B., Tuo, W., Kwok, O.C., Dubey, J.P. 2015. Characterization of the Eimeria maxima sporozoite surface protein IMP1. Veterinary Parasitology. doi: 10.1016/jvetpar2015.05.009.Interpretive Summary: Avian coccidiosis is an important intestinal disease of poultry caused by protozoa in the genus Eimeria.  This disease causes over $ 1 billion in losses worldwide to the poultry industry due to poor weight gain or egg production by infected chickens.  Disease outbreaks are controlled by either medication of poultry feed with anticoccidial compounds or by the vaccination of newly hatched chicks with low doses of Eimeria oocysts.  An alternative approach to preventing coccidiosis is being sought because of decreased efficacy of drugs, increased regulation to remove antibiotics from animal feed, and the lack of uniformity of vaccination such that many chicks are not immunized against infection.  The present study characterizes a potential recombinant Eimeria protein, named E. maxima IMP1, that was produced by isolating the IMP1 gene and inserting it into harmless Escherichia coli.  This cloning step allowed us to show that the protein is on the surface of the parasite, and that the protein is present in tissue of chickens infected with E. maxima.  This data confirms the importance of IMP1 in E. maxima infection, and may point to the possible use of IMP1 as a subunit vaccine against avian coccidiosis.
                                Technical Abstract: The purpose of this study was to characterize Eimeria maxima immunoprotective protein IMP1 that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens.  RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12 hr of sporulation with a considerable downregulation thereafter.  Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence.  The E. maxima IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors.  Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence.  Antisera specific for recombinant E. maxima IMP1 (rEmaxIMP1) identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting.   Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of both E. maxima sporozoites, with negligible staining of merogonic stages.  Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 hr post-infection, and negligible staining thereafter.  The surface locale and expression of IMP1 during early stages of in vivo development may explain in part the immunoprotective effect of this protein against E. maxima infection.


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