Standard Poodles suffer from a long list of autoimmune diseases including immune mediated hemolytic anemia, immune mediated thrombocytopenia, Evan’s syndrome, immune pancytopenia, chronic thyroiditis, temporal-mandibular myositis, and chronic active hepatitis. However, the two most vexing autoimmune disorders are sebaceous adenitis (SA) and Addison’s disease (AD). There has been a general belief that SA and AD entered the breed as a result of extensive inbreeding starting in the middle of the twentieth century that involved a small group of founders that produced show winning offspring. These offspring and their descendants were widely used by Standard Poodle breeders in North America and exported to the UK, Scandinavia, Australia Continental Europe. This artificial midcentury bottleneck (MCB) has created a severe imbalance and probable loss of genetic diversity. Using genetic tests based on 33 genome-wide and seven dog leukocyte antigen (DLA) class I and II short tandem repeat (STR) markers, we were able to study genetic diversity in poodles from the USA, Canada and Europe. Standard Poodles from all of these geographic regions were closely related, indicating a considerable ongoing transoceanic exchange of dogs. Although Standard Poodles still possess considerable total diversity, 70 % of this diversity resides in only 30 % of the population. This imbalance in diversity was both evident across the genome as well as in the DLA class I and II regions, the latter often associated with autoimmune disease. SA and AD entered the breed through different lines and at different times, but the traits underlying these diseases were more likely ancestral in many dog breeds and inadvertently concentrated as a result of the MCB. The DLA was not strongly implicated in either SA or AD, although several less common haplotypes conferred a moderate degree of risk or protection for one or the other disease, and dogs with minor DLA haplotypes were less prone to SA and AD than dogs with major haplotypes. It may be possible to re-distribute the genetic diversity in the breed with judicious mate selection based on genetic testing over a number of generations. Such an undertaking should reduce the incidence of deleterious simple recessive traits and complex genetic disorders such as autoimmune disease. The emphasis of mate selection should be on maximizing genetic differences by augmenting information gained from pedigrees with genetic tests that more accurately measure genetic diversity across the genome. This study also documented the current use of Miniature Poodles as outcrosses, which are genetically distinct and relatively free of SA and AD.

The objective of this study was to determine the influence of inbreeding and specific STR-associated DLA class I and II haplotypes on the incidence of two common autoimmune diseases in Standard Poodles, SA and AD. As such, it is essentially the story of the MCB and how events in both North America and Europe during and after WWII have influenced genetic diversity and certain health problems in Standard Poodles around the world. The study used both pedigrees and DNA-based genetic markers across the genome and in the DLA class I and II regions to investigate the effects of the MCB on genetic diversity and on health. Classical genetic measurements such as Ho and He for Standard Poodles were comparable to those of many other pure breeds [24], but lower than outbred Landrace-type dogs such as Jack Russell Terriers [24] and indigenous village dogs from the Middle East, SE Asia and Pacific regions [16, 23, 26]. Standard Poodles from the USA, Canada and Europe showed minor genetic differences, supporting a considerable transoceanic exchange of dogs. These findings confirmed observations reported in an earlier study of Standard Poodles from these regions [7]. In order to better define genetic diversity, allele numbers and frequency data was used to compute IR values and construct PCoA plots. DLA class I and II haplotype frequencies were used to interrogate diversity in the DLA and risk associations with SA and AD, while pedigrees were used to better define the evolution of the MCB and its effect on genetic diversity and disease.

IR has been widely used in studies that have investigated the relationship between inbreeding and fitness. For instance, studies have shown fitness (reproduction, survival) to be inversely correlated to IR in long-finned pilot whales, grey seals and wandering albatross [27]. Sea lions with high IR values were more likely to suffer from a variety of infections than healthy animals that had suffered a sudden traumatic death [28]. IR was also linked to greater reproductive success in male Antarctic fur seals [29] and improved pre-weaning pup survival in grey seals [30]. Banks and colleagues [31] used IR values based on nine genomic STR markers, as well as a STR loci linked to DAB1 of the major histocompatibility complex, to study the role of heterozygosity on survival and endoparasitism of mountain brushtail possums in Australia. Forstmeier and colleagues [32] demonstrated a positive correlation between lack of heterozygosity as measured by IR and fitness related traits in zebra finches. Remarkably, a small panel of 11 STRs in the latter study produced equally strong results as the large panel of 1359 SNP markers, supporting the continuing value of STRs in population genetics in an era dominated by SNPs.

A genetic profile of Standard Poodles from across the world was created using IR as a measure of heterozygosity and inbreeding. The average Standard Poodles appeared to be reasonably unrelated based on IR values from across the breed. However, when the IR curve was adjusted for diversity lost during breed evolution using village dog allele frequencies as a gold standard [16], average contemporary Standard Poodles were more akin to offspring of village dog parents that were full siblings. This loss of breed diversity has probably occurred in both slow and sudden steps. A “natural” loss of diversity undoubtedly started even earlier when the forefathers of the breed began to deviate from the native (landrace-type) dogs of their regions in Europe as they took on more human selected form and function. The next artificial genetic bottleneck occurred in 1874 in England when the breed registry was established. However, the founding population was apparently quite large and involved dogs with a wide range of form and function and from several geographic regions. It can be surmised that the original registered poodles possessed considerable genetic diversity and that the greatest loss of diversity occurred over the following century. WWII caused many European bloodlines to disappear, especially in countries such as England and Germany where Standard Poodles were prominent. Size limits were also subsequently applied by some registries in Europe, eliminating bloodlines with dogs exceeding the size standard. The loss of breeding lines and ease of international travel also encouraged an influx of Standard Poodles from North America, which were products of the MCB and a better fit to the desired breed type of the time. The fall of the Iron Curtin had a similar effect, but rather than providing a rich source of diversity that had been long insulated from the rest of Europe and North America, breeders from the Iron Curtin countries rushed to replace their indigenous bloodlines with what they perceived as more elegant Western dogs.

Although pedigrees of sufficient depth are helpful in documenting the various artificial genetic bottlenecks of the last 150 years, they do not provide an accurate picture of how these events have affected genetic diversity. Therefore, the purpose of this study was to combine pedigrees and modern DNA-based tests to better define how much the breed has been genetically affected by past events, and in particular the MCB. The effective number of alleles per genomic locus for the breed was low (3.4 alleles/locus) and, on average, 70 % of Standard Poodles tended to share the same two alleles at each locus. This was a portion of what was found in many indigenous village dog populations [16]. This loss of diversity is also reflected by numbers of published maternal and paternal lineages. Standard Poodles descend from one major (82 % of individuals) and 4 minor matrilines and a single patriline [7, 16]. In addition to loss of genetic diversity, a severe imbalance in existing diversity was evident from PCoA and STR-associated DLA class I and II haplotype frequencies. PCoA plots confirmed that a large proportion of the population was more homogeneous with comparatively few genetic outliers. The relatedness of most Standard Poodles was also reflected by a lack of diversity in the STR-associated DLA class I and II regions. The two most common DLA class I haplotypes comprised 45 % of all recognized haplotypes and the four most common made up 90 %. A single DLA class II haplotype was present in a heterozygous or homozygous state in 83 % of the population, which was similar to our earlier study [7].

A conclusion of this study was that inbreeding resulting from artificial genetic bottlenecks such as the MCB resulted in the entrance of autoimmune diseases such as SA and AD into the breed. Genomic markers, when analyzed for % observed heterozygosity, showed that the subpopulation of dogs with SA were less heterozygous than either healthy control dogs or AD dogs. IR values confirmed that dogs with SA were indeed more inbred as a whole than healthy dogs or dogs with AD. However, there was no difference in the healthy and AD populations based on either % observed heterozygosity or IR values. PCoA based on genomic markers placed dogs with both SA and AD in close association with each other and with the main and most inbred population of Standard Poodles. These findings indicated that the genetic traits responsible for SA are not completely fixed in the 70 % of the poodle population that is at greatest risk, while the traits responsible for AD are probably fixed.

Pedigree analysis also linked both SA and AD with the MCB and a rapid rise in COIs starting in the 1960s and peaking in the 1980s. The COIs based on deep pedigrees decreased thereafter, possibly because more and more breeders acted on Armstrong’s admonition to choose mates more carefully to lower COIs [17]. The relatively recent increase in % OEA over the last two decades may reflect attempts to lower the % MCB and % Wycliffe. Although increasing COI was associated with an increased incidence of SA and AD in the population, the effect of recent decreases in COI on these diseases is unknown, as no accurate incidence figures have been compiled. Anecdotally, Standard Poodle breeders believe that the incidence of SA is decreasing in the 2010s, although the situation for AD is more uncertain.

Although pedigree studies indicate that both SA and AD are associated with the MCB, they do not answer the question as to whether the causative traits were ancestral or new. The fact that SA and AD occur so commonly in a number of seemingly unrelated breeds strongly favors an ancestral origin for both disorders [13, 14, 33]. Pedigrees coupled with disease histories also suggest that the genetic traits responsible for SA and AD entered the MCB through different lineages and at different times and that the incidence increased rapidly when the genetic contributions of those ancestors reached a critical threshold or tipping point. In this scenario, the MCB was the force that both disseminated and concentrated the genetic polymorphisms responsible for increased SA and AD susceptibility.

This study also attempted to identify a significant association between specific STR-associated DLA class I or II haplotypes and either SA or AD. Given the number of dogs to be tested, a decision was made to use STRs to determine DLA class I and II haplotypes rather than sequencing. Although, DLA class II haplotypes are easily defined by sequencing polymorphic regions in exon 2 of DQB1, DQA1 and DRB1 genes [34], it does require time and expense, particularly when done on many hundreds of dogs. Identifying DLA class I haplotypes is much more difficult, time consuming and expensive, even with more improved techniques [35]. The GC richness in the DLA88 gene affects the efficiency of priming and amplification and there is a need for extensive cloning. Therefore, there was a distinct advantage in using linked STR markers for both DLA regions. DLA-linked STRs reflect the genetic makeup within its position on the genome, regardless of structural factors that limit PCR amplification and sequencing. As expected, there was strong linkage between specific STR alleles and DLA class I and II haplotypes. Given the strong linkage disequilibrium inherent to the DLA region [6, 7, 16], the occurrence of extended haplotypes between STR-associated DLA class I and II haplotypes was expected. The relatively high incidence of recombination between the DLA class I and II regions, which might not be expected, can be explained by recombination hotspots that exist in the major histocompatibility complex [36].

Several moderate protective and disease susceptibility associations were found between specific DLA Class I haplotypes and SA or AD. Only the STR-associated DLA class I haplotype 1003 was significantly associated with SA and AD at a low to moderate RR of 1.63 and 1.43, respectively. There was also a moderate protective association with having any of the minor class I haplotypes 1008–1057 versus the major haplotypes 1001–1007. At least two DLA class II haplotypes 2004 and 2006, conferred 2.4–2.8 times greater risk for develop AD (p = 0.01) or SA (p = 0.009). Homozygosity among these DLA haplotypes did not further enhance risk, which was also observed in an earlier study of SA in Standard Poodles that interrogated the entire DLA with SNPs [7]. The fact that SA and AD were associated with different DLA class I and II haplotypes supports what is observed in humans, i.e., that predisposition for autoimmune disease may involve many regions of the genome, but the clinical form that it takes is often determined by the HLA [20].

Although certain DLA class I and II haplotypes conferred an increased risk for AD and SA, or were protective, they only occurred in 3–9 % of the population. There is some question, therefore, as to the actual nature of DLA class II haplotype associations with various autoimmune diseases in dogs [37]. The entire DLA region is under strong linkage disequilibrium [6, 7, 16] and inbreeding can result in disproportionately fewer and fewer DLA haplotypes as more and more diversity is lost. If, as the present study demonstrates, autoimmune disease becomes more prevalent in the most inbred portions of the population, it follows that certain DLA class I and II haplotypes could undergo either real or inadvertent positive selection and become either a cause or marker of autoimmunity. If real, associations based solely on relative haplotype frequencies in healthy and diseased dogs should be confirmable by genome wide association studies (GWAS). No significant association with SA was found in the DLA region of Standard Poodles using single nucleotide polymorphisms (SNPs) from GWAS [7]. In contrast, the strong DLA class II association observed in Pug dogs with NME was confirmed by a significant hits in the DLA class II region by genome wide association studies (GWAS) using both STRs [38] and SNPs [39]. GWAS also implicated the DLA in pancreatic acinar atrophy in German Shepherd dogs [13] and later confirmed by sequencing [40]. In contrast, a DLA class II haplotype association in anal furunculosis in this same breed could not be confirmed by GWAS [40]. Genome wide association studies have also failed to confirm DLA association in lupus-like disease in Nova Scotia Duck Tolling Retrievers [8].

Controversy has existed from the onset of closed registries over the effects of inbreeding in dogs, as it has been with humans, other animals and even plants. However, inbreeding has occurred to some degree in dogs since a species of small wolves attached their evolutionary fate to humans as long as 40,000 years ago [41]. This initial attachment undoubtedly involved genetic adaptation of a Darwinian nature, and even though environmental pressures led to non-random selection to increase fitness for a new life-style, it was natural and not man-made. Positive selection undoubtedly accelerated as dogs crossed the threshold from camp followers to camp participants and was more likely to involve human rather than natural selection [16]. Further non-random and human driven selection can be attributed to the Neolithic period when people settled into an agricultural and pastoral lifestyle and dogs integrated with village life [42]. Dogs from this Neolithic expansion became the village dogs and landraces of the world, and individuals from these populations were selected for their abilities to perform specific tasks, including hunting in various forms, guardian of home or flock, and simple companionship. The effect of this gradual inbreeding over thousands of years on the health of Canis familiaris has gone largely unappreciated. However, it is evidenced by the wide range of heritable disorders that plague modern dogs, regardless of whether they are random-, mixed-, or pure-bred [43]. Although these ancient heritable diseases are troublesome, they have become more or less accepted. An even more profound effect of inbreeding has occurred since the Victorian era and the creation of registries that codified specific breeds and closed populations from outside genetic introgressions. This subsequent period of intense inbreeding has not only increased the incidence of ancient diseases, but it has also led to a great increase in simple Mendelian diseases. These simple, and almost exclusively recessive, deleterious traits are much more likely to be seen in purebred dogs, and many are breed specific. Some of these recessive mutations are also ancient, but never of consequence given their previously low frequency in the population. Many of these deleterious recessive traits are new. In either case, inadvertent positive selection has brought them to the forefront. They often occur in complex genetic pathways affecting organs such as eyes, heart/blood, muscle, or nervous system with some regularity [44]. They have been documented in 500 known dog breeds and new ones are published and catalogued monthly [45]. This large array of continually evolving genetic diseases has even promoted the use of purebred dogs for studies of corresponding diseases in humans [46].

One of the objectives of this study was to provide Standard Poodle breeders with the genetic information required to manage diversity within the breed and improve its health. This study implicates past and present inbreeding as a major risk factor for diseases such as SA and AD. Therefore, increasing genetic diversity across the breed should decrease the incidence of these diseases, as well as other heritable disorders. Given the long history of transoceanic interbreeding of Standard Poodles, geographic separation is not a reliable predictor or source of genetic diversity. Genetic diversity can be measured by both pedigrees and genetic tests based on DNA. Pedigrees are valuable in calculating indices such as % MCB, tracing ancestors that may have been sources of heritable diseases, or calculating COIs. However, they must be deep enough to span important genetic bottlenecks or founder effects. Moreover, pedigrees measure theoretical genetic contributions of ancestors, while tests for genetic markers measure the actual contributions. Therefore, tests based on genetic markers should be used to complement and enhance pedigree data. The primary goal of genetic testing should be to identify genome-wide diversity and to use this information to increase, rebalance or introduce new diversity. Maintaining genetic diversity in the DLA region is also important for balancing self and non-self antigen recognition, although assessing and regulating genome-wide diversity purely through DLA markers is genetically flawed. Standard Poodle breeders may have several strategies to confront their genetic diversity problems [47]. The simplest strategy may be to rebalance the genetic diversity that still exists across the breed. This can be done by increasing the contribution of genetic outliers, which are a minority of the population but contain a majority of the genetic diversity. Although this study tried to identify as much existing diversity among Standard Poodles as possible, more genetic diversity may still exist and should be sought out. Diversity could also be increased by bringing in entirely new blood, such as the outcrossing of Standard Poodles with Miniature Poodles. Although Miniature Poodles are genetically distinct from Standard Poodles [54], poodles are registered by size and such crosses are allowed by the AKC and some other registries but are not widely accepted. Although Miniature Poodles can add a significant additional pool of genetic diversity to Standard Poodles, there are also similar breeds that could be used for outcrossing. However, this must be done with great care to preserve and distribute new diversity and to prevent the introduction of new heritable disease traits. Finally, research must continue to identify specific causes and genetic markers for heritable diseases that currently affect the breed’s health, such as SA and AD.

We demonstrated that the incidence of both Addison’s disease and sebaceous adenitis in Standard Poodles increased as a result of an artificial genetic bottleneck involving a small group of show-winning founders from the mid-twentieth century. This bottleneck led to inbreeding over two decades with the results that 50–60 % of an average Standard Poodles ancestry can be traced to a few lines. We conclude that a number of ancestral traits associated with autoimmunity, some common to both SA and AD and some unique to each, were concentrated by inadvertent positive selection during close linebreeding for show winning form.