Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in “humanized” HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

Studies have shown in humans and other species that the major histocompatibility complex class I (MHC-I) region is involved at a number of levels in the establishment and maintenance of pregnancy. The aim of this study was to characterize how a bovine nonclassical MHC-I gene (NC1) is regulated. Initial serial deletion experiments of a 2-kb fragment of the NC1 promoter identified regions with positive regulatory elements in the proximal promoter and evidence for a silencer module(s) further upstream that cooperatively contributed to constitutive NC1 expression. The cytokines interferon tau (IFNT), interferon gamma (IFNG), and interleukin 4 (IL4) significantly increased luciferase expression in NC1 promoter reporter constructs and endogenous NC1 mRNA levels in a bovine endometrial cell line. In addition, IFNG, IL3, IL4, and progesterone significantly increased Day 7 bovine blastocyst NC1 mRNA expression when supplemented during in vitro embryo culture. Site-directed mutagenesis analysis identified a STAT6 binding site that conferred IL4 responsiveness in the NC1 proximal promoter. Furthermore, methylation treatment of the proximal promoter, which contains a CpG island, completely abrogated constitutive NC1 expression. Overall, the findings presented here suggest that constitutive NC1 expression is regulated positively by elements in the proximal promoter, which are further controlled by upstream silencer modules. The promoter is responsive to IFNT, IFNG, and IL4, suggesting possible roles for these cytokines in bovine preimplantation embryo survival and/or maternal-fetal tolerance. Our studies also suggest that methylation of the proximal promoter, in particular, could play a significant role in regulating NC1 expression.

An in vitro assay was used to assess the efficacy of the proprietary pyrethroid insecticide cypermethrin applied to horses (Deosect spray, 5.0%, w/v Fort Dodge Animal Health) against the biting midge Culicoides nubeculosus (Meigen) (Diptera: Ceratopogonidae) and the mosquitoes Aedes aegypti Linneaus and Culex quinquefasciatus Say (Diptera: Culicidae). Hair was collected from the back, belly and legs of the horses immediately prior to treatment and 7, 14, 21, 28 and 35 days after treatment, and also from untreated controls. In laboratory assays groups of 10 adult female C. nubeculosus, Ae. aegypti or C. quinquefasciatus were exposed to 0.5 g of hair for 3 min. In all cases, little or no mortality was observed in insects kept in contact with the pre-treatment samples or the untreated controls. With post-treatment samples for C. nubeculosus, mortality was close to 80% 7 days after treatment and then declined gradually; mean mortality was still at around 50% for hair collected 35 days after treatment. In general, Ae. aegypti and C. quinquefasciatus appeared to be less susceptible to cypermethrin than C. nubeculosus and the attenuation of the toxic effect declined more quickly with time after treatment. There were differences in the toxicity of hair from different body regions, with hair from the back consistently inducing the highest mortality and hair from the legs the lowest; this effect was more pronounced for C. nubeculosus than Ae. aegypti or C. quinquefasciatus. The results demonstrate the potential for topical insecticide treatment to offer protection to horses against biting flies; but highlight the major differences that exist in susceptibility between different insect species.