The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

This study examines genetic diversity among 102 registered English Bulldogs used for breeding based on maternal and paternal haplotypes, allele frequencies in 33 highly polymorphic short tandem repeat (STR) loci on 25 chromosomes, STR-linked dog leukocyte antigen (DLA) class I and II haplotypes, and the number and size of genome-wide runs of homozygosity (ROH) determined from high density SNP arrays. The objective was to assess whether the breed retains enough genetic diversity to correct the genotypic and phenotypic abnormalities associated with poor health, to allow for the elimination of deleterious recessive mutations, or to make further phenotypic changes in body structure or coat. An additional 37 English bulldogs presented to the UC Davis Veterinary Clinical Services for health problems were also genetically compared with the 102 registered dogs based on the perception that sickly English bulldogs are products of commercial breeders or puppy-mills and genetically different and inferior. Four paternal haplotypes, with one occurring in 93 % of dogs, were identified using six Y-short tandem repeat (STR) markers. Three major and two minor matrilines were identified by mitochondrial D-loop sequencing. Heterozygosity was determined from allele frequencies at genomic loci; the average number of alleles per locus was 6.45, with only 2.7 accounting for a majority of the diversity. However, observed and expected heterozygosity values were nearly identical, indicating that the population as a whole was in Hardy-Weinberg equilibrium (HWE). However, internal relatedness (IR) and adjusted IR (IRVD) values demonstrated that a number of individuals were the offspring of parents that were either more inbred or outbred than the population as a whole. The diversity of DLA class I and II haplotypes was low, with only 11 identified DLA class I and nine class II haplotypes. Forty one percent of the breed shared a single DLA class I and 62 % a single class II haplotype. Nineteen percent of the dogs were homozygous for the dominant DLA class I haplotype and 42 % for the dominant DLA class II haplotype. The extensive loss of genetic diversity is most likely the result of a small founder population and artificial genetic bottlenecks occurring in the past. The prominent phenotypic changes characteristic of the breed have also resulted in numerous large runs of homozygosity (ROH) throughout the genome compared to Standard Poodles, which were phenotypically more similar to indigenous-type dogs. English bulldogs have very low genetic diversity resulting from a small founder population and artificial genetic bottlenecks. Although some phenotypic and genotypic diversity still exists within the breed, whether it is sufficient to use reverse selection to improve health, select against simple recessive deleterious traits, and/or to accommodate further genotypic/phenotypic manipulations without further decreasing existing genetic diversity is questionable.

Sebaceous adenitis (SA) and Addison’s disease (AD) increased rapidly in incidence among Standard Poodles after the mid-twentieth century. Previous attempts to identify specific genetic causes using genome wide association studies and interrogation of the dog leukocyte antigen (DLA) region have been non-productive. However, such studies led us to hypothesize that positive selection for desired phenotypic traits that arose in the mid-twentieth century led to intense inbreeding and the inadvertent amplification of AD and SA associated traits. This hypothesis was tested with genetic studies of 761 Standard, Miniature, and Miniature/Standard Poodle crosses from the USA, Canada and Europe, coupled with extensive pedigree analysis of thousands more dogs. Genome-wide diversity across the world-wide population was measured using a panel of 33 short tandem repeat (STR) loci. Allele frequency data were also used to determine the internal relatedness of individual dogs within the population as a whole. Assays based on linkage between STR genomic loci and DLA genes were used to identify class I and II haplotypes and disease associations. Genetic diversity statistics based on genomic STR markers indicated that Standard Poodles from North America and Europe were closely related and reasonably diverse across the breed. However, genetic diversity statistics, internal relatedness, principal coordinate analysis, and DLA haplotype frequencies showed a marked imbalance with 30 % of the diversity in 70 % of the dogs. Standard Poodles with SA and AD were strongly linked to this inbred population, with dogs suffering with SA being the most inbred. No single strong association was found between STR defined DLA class I or II haplotypes and SA or AD in the breed as a whole, although certain haplotypes present in a minority of the population appeared to confer moderate degrees of risk or protection against either or both diseases. Dogs possessing minor DLA class I haplotypes were half as likely to develop SA or AD as dogs with common haplotypes. Miniature/Standard Poodle crosses being used for outcrossing were more genetically diverse than Standard Poodles and genetically distinguishable across the genome and in the DLA class I and II region. Ancestral genetic polymorphisms responsible for SA and AD entered Standard Poodles through separate lineages, AD earlier and SA later, and were increasingly fixed by a period of close linebreeding that was related to popular bloodlines from the mid-twentieth century. This event has become known as the midcentury bottleneck or MCB. Sustained positive selection resulted in a marked imbalance in genetic diversity across the genome and in the DLA class I and II region. Both SA and AD were concentrated among the most inbred dogs, with genetic outliers being relatively disease free. No specific genetic markers other than those reflecting the degree of inbreeding were consistently associated with either disease. Standard Poodles as a whole remain genetically diverse, but steps should be taken to rebalance diversity using genetic outliers and if necessary, outcrosses to phenotypically similar but genetically distinct breeds.

Laboratory cats were infected with a serotype I cat-passaged field strain of FIP virus (FIPV) and peritoneal cells harvested 2–3 weeks later at onset of lymphopenia, fever and serositis. Comparison peritoneal cells were collected from four healthy laboratory cats by peritoneal lavage and macrophages predominated in both populations. Differential mRNA expression analysis identified 5621 genes as deregulated in peritoneal cells from FIPV infected versus normal cats; 956 genes showed > 2.0 Log2 Fold Change (Log2FC) and 1589 genes showed < −2.0 Log2FC. Eighteen significantly upregulated pathways were identified by InnateDB enrichment analysis. These pathways involved apoptosis, cytokine–cytokine receptor interaction, pathogen recognition, Jak-STAT signaling, NK cell mediated cytotoxicity, several chronic infectious diseases, graft versus host disease, allograft rejection and certain autoimmune disorders. Infected peritoneal macrophages were activated M1 type based on pattern of RNA expression. Apoptosis was found to involve large virus-laden peritoneal macrophages more than less mature macrophages, suggesting that macrophage death played a role in virus dissemination. Gene transcripts for MHC I but not II receptors were upregulated, while mRNA for receptors commonly associated with virus attachment and identified in other coronaviruses were either not detected (APN, L-SIGN), not deregulated (DDP-4) or down-regulated (DC-SIGN). However, the mRNA for FcγRIIIA (CD16A/ADCC receptor) was significantly upregulated, supporting entry of virus as an immune complex. Analysis of KEGG associated gene transcripts indicated that Th1 polarization overshadowed Th2 polarization, but the addition of relevant B cell associated genes previously linked to FIP macrophages tended to alter this perception.

Previous studies documented the problem of inbreeding among Italian Greyhounds (IG) from the USA and its possible role in a multiple autoimmune disease syndrome. The present study is an extension of these earlier experiments and had two objectives: 1) to identify pockets of additional genetic diversity that might still exist among IG from the USA and Continental Europe, and 2) to determine how loss of genetic diversity within the genome and in the dog leukocyte antigen (DLA) complex relates to the problem of autoimmune disease in IG from the USA. Genetic testing was conducted using 33 short tandem repeat (STR) loci across 25 chromosomes and 7 STR loci that associated with specific dog leukocyte antigen (DLA) class I and II haplotypes. Standard genetic assessment tests based on allele frequencies and internal relatedness (IR) were used as measures of breed-wide and individual heterozygosity. The results of these tests demonstrated that IG from the USA and Continental Europe belonged to a single breed but were genetically distinguishable by genomic allele frequencies, DLA class I and II haplotypes, and principal coordinate analysis (PCoA). In the second part of the study, 85 IG from the USA that had suffered various autoimmune disorders (case) and 104 healthy dogs (control) of comparable age were studied for genetic associations with disease. Case dogs were found to be significantly more homozygous in the DLA regions than control dogs. Principal coordinate analysis did not differentiate case from control populations. No specific STR-associated DLA-class I or II haplotype was associated with increased autoimmune disease risks. Reasons for the loss of genetic diversity and increased homozygosity among IG from the USA were studied using registration data and deep pedigrees. The breed in the USA started from a small number of founders from Europe and has remained relatively isolated and small in numbers, limiting breeding choices especially in the period before modern transportation and artificial insemination. An additional cause of lost diversity and increased homozygosity has been the influence of famous sires and their show-winning progeny. The most influential of these sires was Ch. Dasa’s King of the Mountain (King) born in 1978. Virtually all contemporary IG from the USA have King at least once in 10 generation pedigrees and 18 % of the genome of contemporary IG from the USA is shared with King. It was concluded that artificial genetic bottlenecks have concentrated numerous genetic polymorphisms responsible for autoimmune disease and that these risk factors did not originate in a specific individual or bloodline of the breed. Rather, they were of ancestral origin in both purebred and random bred dogs and inherited by descent. Italian Greyhound breeders in the USA have several options to improve breed health: 1) breed against homozygosity within the genome and in the DLA region, 2) avoid breeding dogs that have suffered an autoimmune disorder, 3) increase diversity by incorporating the genetic differences that exist in IG from Continental Europe, or 4) outcross to other small sighthound breeds. The latter two approaches must be undertaken with care to avoid introduction of new deleterious traits and to maximize retention and dissemination of new genetic diversity.