Diversity of major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes and their interactions in domestic horses.

The immunogenome is the part of the genome that underlies immune mechanisms and evolves under various selective pressures. Two complex regions of the immunogenome, major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes, play an important role in the response to selective pressures of pathogens. Their importance is expressed by their genetic polymorphism at the molecular level, and their diversity associated with different types of diseases at the population level. Findings of associations between specific combinations of MHC/NKR haplotypes with different diseases in model species suggest that these gene complexes did not evolve independently. No such associations have been described in horses so far. The aim of the study was to detect associations between MHC and NKR gene/microsatellite haplotypes in three horse breed groups (Camargue, African, and Romanian) by statistical methods; chi-square test, Fisher's exact test, Pearson's goodness-of-fit test and logistic regression. Associations were detected for both MHC/NKR genes and microsatellites; the most significant associations were found between the most variable KLRA3 gene and the EQCA-1 or EQCA-2 genes. This finding supports the assumption that the KLRA3 is an important receptor for MHC I and that interactions of these molecules play important roles in the horse immunity and reproduction. Despite some limitations of the study such as low numbers of horses or lack of knowledge of the selected genes functions, the results were consistent across different statistical methods and remained significant even after overconservative Bonferroni corrections. We therefore consider them biologically plausible.

Newly identified variability of the antigen binding site coding sequences of the equine major histocompatibility complex class I and class II genes.

The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in three genetically distinct horse breeds. A total of 116 allelic variants were found in the MHC class I genes Eqca-1, Eqca-2, Eqca-7 and Eqca-Ψ, 112 of which were novel. The MHC class II DRA locus was confirmed to comprise five exon 2 alleles, and no new sequences were observed. Additional variability in terms of 15 novel exon 2 alleles was identified in the DQA1 locus. Extensive overall variability across the entire MHC region was confirmed by an analysis of MHC-linked microsatellite loci. Both diversifying and purifying selection were detected within the MHC class I and II loci analyzed.

The natural cytotoxicity receptor genes in the family Felidae.

Natural killer (NK) cells belong to the innate immune system. The germline-encoded natural killer cell receptors represent activating and inhibitory receptors regulating multiple NK cell activities. The natural cytotoxicity receptors (NCRs) are activating natural cytotoxicity triggering receptors 1, 2, and 3 (NKp46, NKp44, and NKp30), encoded by the genes NCR1, NCR2, and NCR3, respectively. NCRs may be expressed in different cell types engaged in mechanisms of innate and adaptive immunity. The family Felidae, comprising the domestic cat and a wide variety of free-ranging species represents a well-suited model for biomedical and evolutionary studies. We characterized the NCR1, NCR2, and NCR3 genes in a panel of felid species. We confirmed the presence of potentially functional genes NCR1, NCR2, and NCR3 in all species. All three genes are conserved within the family and are similar to other phylogenetically related mammalian families. The NCR1 and NCR2 phylogenetic trees based on both nucleotide and protein sequences corresponded to the current zoological taxonomy, with some exceptions suggesting effects of different selection pressures in some species. Highly conserved NCR3 sequences did not allow a robust phylogenetic analysis. Most interspecific differences both at the nucleotide and protein level were found in NCR2. Within species, the most polymorphic CDS was detected in NCR1. Selection analyses indicated the effects of purifying selection on individual amino acid sites in all three genes. In stray cats, a rather high intraspecific diversity was observed.

Shedding persistency and intensity patterns of feline coronavirus (FCoV) in feces of cats living in breeding catteries in the Czech Republic.

Infection with feline coronavirus (FCoV) is a major problem in multiple-cat households, where many cats are kept together in a small space such as catteries and shelters. Sixty cats from 19 breeding catteries included in the study were evaluated for their shedding persistency and intensity patterns using qPCR identification of FCoV in feces. Cats were identified based on shedding persistency as non-shedders (NS) if all four samples negative, intermittent shedders (IS) when at least one positive and one negative sampling followed by another positive sampling, persistent shedders (PS) if all four samples positive and shedders with unclear status (US) if the shedding patterns could not be determined based on only 4 samples. There were 11 NS (18%), 15 IS (25%) and 15 PS (25%) and in 19/60 cats (32%), the shedding patterns could not be determined based only on four samplings. The intensity of shedding was evaluated based on the total number of FCoV particles shed during the 12 months of the study. There were 11 non-shedders (18%), 2 very low intensity shedders (3%), 9 low intensity shedders (15%), 25 medium intensity shedders (42%) and 13 high intensity shedders (22%). Intermittent shedders were shedding significantly lower FCoV particles compared to the persistent shedders (p = 0.0082). Permanent shedders represent the most important source of FCoV infection in multi-cat households and identifying permanent shedders in is the key to minimize the viral load in the environment to control FCoV in a shelters and breeding catteries.

The Population Diversity of Candidate Genes for Resistance/Susceptibility to Coronavirus Infection in Domestic Cats: An Inter-Breed Comparison.

Feline coronavirus (FCoV) is a complex pathogen causing feline infectious peritonitis (FIP). Host genetics represents a factor contributing to the pathogenesis of the disease. Differential susceptibility of various breeds to FIP was reported with controversial results. The objective of this study was to compare the genetic diversity of different breeds on a panel of candidate genes potentially affecting FCoV infection. One hundred thirteen cats of six breeds were genotyped on a panel of sixteen candidate genes. SNP allelic/haplotype frequencies were calculated; pairwise FST and molecular variance analyses were performed. Principal coordinate (PCoA) and STRUCTURE analyses were used to infer population structure. Interbreed differences in allele frequencies were observed. PCoA analysis performed for all genes of the panel indicated no population substructure. In contrast to the full marker set, PCoA of SNP markers associated with FCoV shedding (NCR1 and SLX4IP) showed three clusters containing only alleles associated with susceptibility to FCoV shedding, homozygotes and heterozygotes for the susceptibility alleles, and all three genotypes, respectively. Each cluster contained cats of multiple breeds. Three clusters of haplotypes were identified by PCoA, two clusters by STRUCTURE. Haplotypes of a single gene (SNX5) differed significantly between the PCoA clusters.

Candidate Gene Markers Associated with Fecal Shedding of the Feline Enteric Coronavirus (FECV).

The Feline coronavirus (FCoV) can cause a fatal disease, the Feline Infectious Peritonitis. Persistent shedders represent the most important source of infection. The role of the host in FCoV fecal shedding is unknown. The objective of this study was to develop gene markers and to test their associations with FCoV shedding patterns. Fecal samples were taken from 57 cats of 12 breeds on the day 0 and after 2, 4 and 12 months. Variation from persistent and/or high-intensity shedding to no shedding was observed. Thirteen immunity-related genes were selected as functional and positional/functional candidates. Positional candidates were selected in a candidate region detected by a GWAS analysis. Tens to hundreds of single nucleotide polymorphisms (SNPs) per gene were identified using next generation sequencing. Associations with different phenotypes were assessed by chi-square and Fisher's exact tests. SNPs of one functional and one positional candidate (NCR1 and SLX4IP, respectively) and haplotypes of four genes (SNX5, NCR2, SLX4IP, NCR1) were associated with FCoV shedding at pcorected < 0.01. Highly significant associations were observed for extreme phenotypes (persistent/high-intensity shedders and non-shedders) suggesting that there are two major phenotypes associated with different genotypes, highly susceptible cats permanently shedding high amounts of viral particles and resistant non-shedders.