γδ T lymphocytes are recruited into the inflamed uterus of bitches suffering from pyometra.

Very little is known about the occurrence of immune system cells in the canine uterus. The aim of this study was to generate information about lymphocyte subsets that are present in the healthy canine uterus and that are recruited under inflammatory conditions caused by pyometra. Using immunohistochemistry and flow cytometry, a significant influx of γδ T lymphocytes was found in pyometra samples mainly due to recruitment of γδ(+)/CD8(-) T lymphocytes. The relative expression of genes encoding selected cytokines/chemokines was evaluated in samples from healthy and pyometra-affected uteri. Expression of pro-inflammatory cytokines (including IL-1ß, TNF-α, IL-8, IL-17 and IFN-γ) and chemokines (including CXCL10, CCL4 and CCL5) was upregulated in pyometra samples confirming the presence of inflammation. In contrast, the expression of the homeostatic chemokine CCL25 and of the anti-inflammatory cytokine IL-10 was downregulated and unchanged, respectively.

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils.

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1ß and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.

Experimental oral and ocular Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has a wide host distribution, but primarily affects rabbits. The aim of this study was to characterize both the cell-mediated and the antibody response in rabbits after experimental infection using 2 different infection routes: oral and ocular. SPF rabbits were infected with low (10³ spores) and high (107 spores) infection doses. Monitored parameters included clinical signs, detection of spores in urine, antibody response detected with ELISA, and cell-mediated immunity detected by antigen-driven lymphocyte proliferation. At week 13 post-infection, half of the rabbits in each group were suppressed by intramuscular administration of dexamethasone. At week 18 post-infection, animals were euthanized. Clinical signs were mild with exacerbation after immunosuppression. Spores in urine and antigen-specific cell-mediated immunity were detected from weeks 5 and 4 post-infection, respectively. Specific IgM was detected 1 week after infection, and IgG antibodies followed 1 week later in rabbits infected with the high dose. Immunological responses were dose dependent. The authors can conclude that both oral and ocular experimental infection with E. cuniculi resulted in an immune response of the infected animals. Rabbits could be used as an experimental model for the study of ocular microsporidiosis.

Single nucleotide polymorphisms of interleukin-1 beta related genes and their associations with infection in the horse.

In previous work, we found significant associations of horse chromosome 15 (ECA15) microsatellite markers HMSO1 and HTG06 with two horse infections, Rhodococcus equi and Lawsonia intracellularis, respectively. Interleukin-1 beta subunit and interleukin-1 receptor antagonist encoding genes (IL1B and IL1RN) could be considered as candidate genes underlying the associations reported. Therefore, we identified single nucleotide polymorphisms (SNPs) within three interleukin-1 beta functionally related genes: IL1B, IL1RN and Casp1 (interleukin-1 beta converting enzyme/caspasel encoding gene). Using appropriate restriction fragment length polymorphism (PCR-RFLP) and/or single strand conformation polymorphism (PCR-SSCP) markers, their associations with the two infections by genotyping foals from the original study were tested. In addition, the physical localization of one of the two closely located genes, IL1RN, was re-assessed by fluorescence in-situ hybridization (FISH). A statistically significant association between an intronic SNP of the Casp1 gene with R. equi infection was found. The IL1RN gene was localized to 15q13-q14 in agreement with its originally reported physical position.

Cytogenetic mapping of immunity-related genes in the domestic horse.

Chromosomal locations of 19 horse immunity-related loci (CASP1, CD14, EIF5A, FCER1A, IFNG, IL12A, IL12B, IL12RB2, IL1A, IL23A, IL4, IL6, MMP7, MS4A2, MYD88, NOS2A, PTGS2, TFRC and TLR2) were determined by fluorescence in situ hybridization. For IFNG and PTGS2, this study is a confirmation of their previously reported position. In addition, microsatellite (HMBr1) was localized in the same region as IFNG. All genes were assigned to regions of conserved synteny and the data obtained in this study enhance the comparative human-horse map. Cytogenetic localization of IL6 to ECA4q14-q21.1 suggested a new breakage point that changes the order of loci compared with HSA7. The map assignments of these loci serve as anchors for other loci and will aid in the search for candidate genes associated with traits in the horse.

Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cepsilon, andFcepsilon R1 alpha.

The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cepsilon encoding the IgE heavy chain molecule and the gene FcepsilonR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5'UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5'UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cepsilon gene, and one SNP was found in the FcepsilonR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.

Characterization of the NRAMP1 (SLC11A1) gene in the horse (Equus caballus L.).

The complete coding cDNA sequence of the horse NRAMP1 (SLC11A1) gene was determined (GenBank accession number AF354445). The nucleotide sequence of the horse NRAMP1 gene is similar to sequences of this gene in other species. The gene contains 15 exons whose total length of 1,635 bp corresponds to 544 amino acids constituting the resulting putative protein. Hydrophobicity profile analysis of the deduced horse NRAMP1 gene product showed a nearly identical structure with the mouse NRAMP1 protein. The gene was found to be located on the short arm of ECA 6p12-13 by fluorescence in situ hybridization (FISH) analysis. Five allelic variants of the 5' untranslated region (UTR) were identified at the nucleotide sequence level. PCR-RFLP polymorphisms for NlaIII, TaqI, MspI and AciI were detected. Four out of five alleles could be detected using TaqI and MspI restriction enzymes. Their haplotype frequencies were different in four genetically distinct horse breeds.

Two bi-allelic single nucleotide polymorphisms within the promoter region of the horse tumour necrosis factor alpha gene.

Primers based on GenBank sequences within the 5' untranslated region (UTR) of the human and horse tumour necrosis factor alpha (TNF-alpha) genes were designed and used to amplify a 522-bp product. Sequencing of five clones derived from five independent PCRs obtained from three different animals of three different breeds (Old Kladruber, Akhal-Teke and Shetland Pony) revealed a high level of sequence identity to the TNF-alpha promoter regions of other species. The existing GenBank horse sequences were confirmed and extended upstream by 230 nucleotides. Based on the sequence obtained, a new horse-specific forward primer was designed to amplify a 213-bp PCR product, which was screened for polymorphism using single-strand conformation polymorphism (SSCP). Three allelic variants of the horse TNF-alpha gene were identified and sequenced (GenBank accession numbers ADF 349558-60). Two single nucleotide polymorphisms explained the existence of the three SSCP alleles detected: C/T and T/C single base pair substitutions at positions 137 and 147, respectively. Differences in allelic frequencies between Old Kladruber and Akhal-Teke breeds were observed.

A second locus and new alleles in the major histocompatibility complex class II (ELA-DQB) region in the horse.

More than two nucleotide sequences of the second exon of the ELA-DQB region retrieved from a single animal and two different sequences isolated from horses homozygous in the major histocompatibility complex (MHC) region by descent indicated the existence of at least two ELA-DQB loci at the genomic level. New alleles detected by polymerase chain reaction single strand conformation polymorphism (SSCP) and defined by nucleotide sequencing of the second exon of the DQB gene(s) were described. Based on the level of nucleotide sharing, at least two groups of alleles were shown to exist. The newly defined alleles belonged preferentially to one of the groups. However, their specific locus assignment was not possible from the data collected. At least one of these alleles was shown to be transcribed. No frame-shift mutations were identified among the new alleles, although one pseudoallele containing a stop codon was identified at the genomic DNA level.

BoLA DYA polymorphism in Czech cattle.

Polymorphism at the BoLA DYA locus was determined in two groups of Czech Black Pied cattle by PCR-RFLP detecting substitutions at nucleotide positions 112 and 219. Animals for this study were nonrandomly selected according to their health status in two BoLA-associated infections: bovine leukosis (n = 59) and Johne's disease (n = 36). A group of noninfected Czech Red Pied cows (n = 37) was used for comparison. The frequencies of DYA alleles and haplotypes were virtually identical in the two selected groups as well as in the infection-free animals. In contrast, distribution of BoLA DRB3.2 alleles differed considerably between the infected groups as expected based on the previously detected associations with BoLA. The results suggest that the polymorphism of the DYA unexpressed gene was not influenced by selecting animals for this study according to their health status.