BoLA-6*01301 and BoLA-6*01302, two allelic variants of the A18 haplotype, present the same epitope from the Tp1 antigen of Theileria parva.

We have recently shown that the BoLA-A18 variant haplotype (BoLA-6*01302) is more prevalent than the BoLA-A18 haplotype (BoLA-6*01301) in a sample of Holstein/Friesian cattle in Kenya. These MHC class I allelic variants differ by a single amino acid polymorphism (Glu97 to Leu97) in the peptide-binding groove. We have previously mapped an 11-mer peptide epitope from the Theileria parva antigen Tp1 (Tp1214-224) that is presented by BoLA-6*01301. Crystal structure data indicates that Glu97 in the MHC molecule plays a role in epitope binding through electro-static interaction with a lysine residue in position 5 of the epitope, which also functions as an additional anchor residue. In contrast to expectations, we demonstrate that the amino acid substitution in BoLA-6*01302 does not divert the CTL response away from Tp1214-224. The two MHC molecules exhibit similar affinity for the Tp1 epitope and can present the epitope to parasite-specific CTLs derived from either BoLA allelic variants. These data confirm that this BoLA polymorphism does not alter Tp1 epitope specificity and that both allelic variants can be used for Tp1 vaccine studies.

A method to discriminate between closely related bovine major histocompatibility complex class I alleles by combining established PCR-SSP assays with RFLPs.

We have developed a polymerase chain reaction-sequence-specific primers-restriction fragment length polymorphism (PCR-SSP-RFLP) method to rapidly differentiate between the A18 and A18 variant (v) BoLA haplotypes and between A14 and A15/A15v BoLA haplotypes in Holstein/Friesian cattle. We used published SSP to PCR amplify BoLA alleles expressed in animals of known haplotype and exposed the amplicons to the restriction enzyme PvuII that was predicted to cut at a unique site in the middle of BoLA-6*01302 (A18v) and BoLA-1*00901 (A15) but not in BoLA-6*01301 (A18) or BoLA-1*02301 (A14) alleles. Whereas the method does not discriminate between the A15 and A15v haplotypes, as the BoLA-1*00902 allele associated with A15v also contains a PvuII site, we are interested in cattle of A18 and A14 haplotype for vaccine related studies. Our results also indicated that the BoLA-6*01302 (A18v) allele is much more abundant than BoLA-6*01301 (A18) in the cattle that we sampled.

Ferrets as a model for morbillivirus pathogenesis, complications, and vaccines.

The ferret is a standard laboratory animal that can be accommodated in most animal facilities. While not susceptible to measles, ferrets are a natural host of canine distemper virus (CDV), the closely related carnivore morbillivirus. CDV infection in ferrets reproduces all clinical signs associated with measles in humans, including the typical rash, fever, general immunosuppression, gastrointestinal and respiratory involvement, and neurological complications. Due to this similarity, experimental CDV infection of ferrets is frequently used to assess the efficacy of novel vaccines, and to characterize pathogenesis mechanisms. In addition, direct intracranial inoculation of measles isolates from subacute sclerosing panencephalitis (SSPE) patients results in an SSPE-like disease in animals that survive the acute phase. Since the advent of reverse genetics systems that allow the targeted manipulation of viral genomes, the model has been used to evaluate the contribution of the accessory proteins C and V, and signalling lymphocyte activation molecule (SLAM)-binding to immunosuppression and overall pathogenesis. Similarly produced green fluorescent protein-expressing derivatives that maintain parental virulence have been instrumental in the direct visualization of systemic dissemination and neuroinvasion. As more immunological tools become available for this model, its contribution to our understanding of morbillivirus-host interactions is expected to increase.