Year of Publication

2014

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Veterinary Science

First Advisor

Dr. Daniel K. Howe

Abstract

Sarcocystis neurona is a protozoan parasite that causes the serious neurologic disease equine protozoal myeloencephalitis (EPM). The life cycle of S. neurona progresses through multiple developmental stages that differ morphologically and molecularly. The S. neurona merozoite surface is covered by multiple related proteins, which are orthologous to the surface antigen (SAG) gene family of Toxoplasma gondii. The SAG surface antigens in T. gondii and another related parasite Neospora caninum are life cycle stage-specific and seem necessary for parasite transmission and persistence of infection. The present research was conducted to explore the gene family of SnSAGs in S. neurona. Specifically, the project identified new SnSAGs in the draft genome sequence of S. neurona and examined the stage-specific expression and potential function of these surface antigens. For the first part of the study, expression of the S. neurona merozoite surface antigens was evaluated in the sporozoite and bradyzoite stages. The studies revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed by sporozoites, while SnSAG5 appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. For the second part of the study, the draft sequence of the S. neurona genome was searched for potential new SnSAGs. Multiple searches revealed sixteen potential new SnSAG genes, and bioinformatic analyses of the sequences revealed characteristics consistent with the SAG gene family. Two of the new SnSAGs, designated SnSAG7 and SnSAG8, have been characterized in detail. The studies showed that SnSAG7 is expressed by the merozoite stage, while SnSAG8 is expressed by the bradyzoite stage. The third part of the study assessed the role of SnSAGs in host cell attachment and/or invasion by S. neurona. Serum neutralization assays using polyclonal serum raised against SnSAG1, SnSAG2, SnSAG3, and SnSAG4 suggested that SnSAG1 and SnSAG4 play a role in host cell attachment and/or invasion; treatment with antibodies against SnSAG2 and SnSAG3 were inconclusive. The information acquired about the stage-specific expression of the SnSAGs, identification of new SnSAG paralogues, and their functional characterization will help to understand the importance of the SnSAG proteins for parasite survival and could lead to improved methods for EPM prevention and/or treatment.

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