**4. Gut microbiome and therapeutic application**

The growing understanding of the microbiome's role in carcinogenesis has allowed the microbiome influence to be linked to the effectiveness of cancer therapies. Microbiome modulation strategies can affect cancer treatment through inactivation or activation of chemotherapeutic agents, modification of immune responses and interference with side effects [72]. This relationship is bilateral, in which the systemic cancer therapy influences gut microbiota, and gut microbiota influences cancer treatment [122]. Recent publications indicate the gut microbiome manipulation as a new treatment tool or to improve the response to cancer therapy. Some of the proposed mechanisms will be discussed below.

#### **4.1 Roles of microbiome in cancer therapy**

#### *4.1.1 Chemotherapy*

Iida et al. (2013) demonstrated that microbiota impairs disruption response of subcutaneous tumors to platinum derived chemotherapeutic agents. Tumor-bearing mice that lacked microbiota showed therapy efficacy reduction, given that microbiota was important for activating the innate immune response [123]. In another study, administration of *Ruminococcus gnavus* (bacterial strain depleted by treatment with cisplatin) was able to partially restore intestinal mucosa integrity and reduce systemic inflammation in mice treated with cisplatin [124]. Results indicate that reconstitution of gut microbiome can help healing intestinal epithelium in patients treated with chemotherapy.

On the other hand, Viaud et al. (2013) demonstrated that gut microbiota helps shape anti-cancer immune response of cyclophosphamide (CTX). Using mouse models, it was demonstrated that cyclophosphamide alters intestine microbiota composition and induces translocation of selected species into secondary lymphoid organs, resulting in Th17 cells maturation promoting an adaptive immune response against tumors [125]. Daillere et al. (2016) identified *Enterococcus hirae* and *Barnesiella intestinihominis* species involved in tumor immunosurveillance during cyclophosphamide therapy; *E. hirae* translocates from gut to lymph nodes inducing Th1 and Th17 responses mandatory for anti-tumor activity of CTX, while *B. intestinihominis* increases systemic Th1 and CD8 + cytotoxic T cells, which were associated with an increase of IFN-y-producing γ δ tumor infiltrating-lymphocytes (TILs) contributing also for anti-tumor CTX effect [126]. Therefore, cyclophosphamide immunomodulatory effects require a functional microbiome.

Chemotherapy efficacy can also be impacted by intratumoral bacteria. Geller et al. (2017) showed, in a colon cancer mouse model, that Gammaproteobacteria can metabolize chemotherapeutic gemcitabine into an inactive form inducing chemotherapy resistance and that this effect was reversed by antibiotic ciprofloxacin. Interestingly, about 76% of human pancreatic ductal adenocarcinomas were positive for bacteria, mainly Gammaproteobacteria [127]. Perhaps the treatment for this tumor type may be improved by adding antibiotics to the chemotherapy.

Chemotherapy-induced diarrhea (CD) is a frequent adverse event in dogs, in which changes in gut microbiota appear to play a key role. A recent study of 60 dogs undergoing chemotherapy supported the administration of smectite, a natural medical clay, widely used in acute diarrhea treatment in humans, as a first-line treatment of CD in dogs. Interestingly, smectite has anti-inflammatory properties to decrease intestinal bacterial translocation and stabilize intestinal microbiome [128]. However, studies associating microbiome and effectiveness of chemotherapy are still scarce in veterinary medicine.

#### *4.1.2 Immunotherapy*

Several studies have shown a complex crosstalk between bacteria and immune host response in the anti-tumor battle. For example, Paulos et al. (2007) reported

**125**

*Small Animals Gut Microbiome and Its Relationship with Cancer*

that total body irradiated mice showed a more efficient anti-tumor response to adoptively transferred tumor-specific CD8+ T cells against melanoma after gut microbial translocation to mesenteric lymph nodes. They observed that the radiation induced the release of microbial LPS and activated innate immune response by TLR4 stimulation and then increased anti-tumor CD8+ T cells, while reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS

There is also evidence that gut microbiome modulates efficacy of immune checkpoint inhibitors (CIs), that are monoclonal antibodies with inhibitory effect to specific receptors on T cells and tumor cells, blocking signaling pathways that negatively modulate immune system, allowing specific T cells to promote destruction of cancer cells [130]. Those receptors include cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 protein (PD-1), and programmed

It was demonstrated that oral administration of Bifidobacterium in mice with melanoma was associated with the same degree of antitumor effects as in those mice that received therapy with PD-L1 antibodies, and the combination of both treatments almost abolished tumor growth [132]. In addition, a report of human gut microbiome metagenomic profiling in 39 metastatic melanoma patients treated with anti-PD1 and/or anti-CTLA-4 immunotherapy identified that those who respond to all types of CIs were enriched for *Bacteroides caccae*, enhancing that microbiota may modulate cancer immunotherapy [133]. These correlations have not

**4.2 Therapeutic manipulation of microbiome and its relevance to cancer** 

Given the increasing evidence on the significant role that microbiome can play in cancer, microbiota modulation represents a new therapeutic potential capable of altering disease development. These therapies aim to change the microbial community associated with dysbiosis for those associated with health. In small animals, microbiome manipulations are often described as part of gastrointestinal diseases treatment [134]. Mainly as an adjuvant treatment for cancer, these interventions and their effectiveness are not well established and have only recently been described in literature. The following will discuss some ways in which microbiome

Prebiotics are specific chemicals, capable of promoting growth of a selective group of bacteria and their specific metabolites and thus modulating microbiota in a beneficial way, which may help on anti-tumor treatment [135]. These are non-digestible or absorbable dietary fibers and include fructans (oligofructose and inulin), nonstarch polysaccharides found in some cereal grains, algae, disaccharides (lactulose), and polysaccharides including fructooligosaccharides (FOS) [22]. According to Villegér and colleagues, the effect of prebiotics depends on the presence of beneficial bacteria in the host's intestines [33]. Thus, the combination of probiotics and prebiotics, known as symbiotic, looks promising. Dietary treatment with inulin or oligofructose has been demonstrated to selectively stimulate growth of specific bacterial taxa and alter SCFA levels within the gut [136]. Moreover, these prebiotics reduced the incidence of mammary tumors in rats, significantly potentiated chemotherapy effects as well as RT [136]. The perioperative administration of symbiotics, probiotics (strains Lactobacillus and Bifidobacterium) and prebiotic

signaling were associated with a decrease of anti-tumor response [129].

*DOI: http://dx.doi.org/10.5772/intechopen.95780*

death-ligand 1 (PD-L1) [131].

yet been demonstrated in dogs.

**therapies response**

can be modified:

*4.2.1 Prebiotics and symbiotics*

#### *Small Animals Gut Microbiome and Its Relationship with Cancer DOI: http://dx.doi.org/10.5772/intechopen.95780*

*Canine Genetics, Health and Medicine*

*4.1.1 Chemotherapy*

inactivation or activation of chemotherapeutic agents, modification of immune responses and interference with side effects [72]. This relationship is bilateral, in which the systemic cancer therapy influences gut microbiota, and gut microbiota influences cancer treatment [122]. Recent publications indicate the gut microbiome manipulation as a new treatment tool or to improve the response to cancer therapy.

Iida et al. (2013) demonstrated that microbiota impairs disruption response of subcutaneous tumors to platinum derived chemotherapeutic agents. Tumor-bearing mice that lacked microbiota showed therapy efficacy reduction, given that microbiota was important for activating the innate immune response [123]. In another study, administration of *Ruminococcus gnavus* (bacterial strain depleted by treatment with cisplatin) was able to partially restore intestinal mucosa integrity and reduce systemic inflammation in mice treated with cisplatin [124]. Results indicate that reconstitution of gut microbiome can help healing intestinal epithelium in

On the other hand, Viaud et al. (2013) demonstrated that gut microbiota helps shape anti-cancer immune response of cyclophosphamide (CTX). Using mouse models, it was demonstrated that cyclophosphamide alters intestine microbiota composition and induces translocation of selected species into secondary lymphoid organs, resulting in Th17 cells maturation promoting an adaptive immune response against tumors [125]. Daillere et al. (2016) identified *Enterococcus hirae* and *Barnesiella intestinihominis* species involved in tumor immunosurveillance during cyclophosphamide therapy; *E. hirae* translocates from gut to lymph nodes inducing Th1 and Th17 responses mandatory for anti-tumor activity of CTX, while *B. intestinihominis* increases systemic Th1 and CD8 + cytotoxic T cells, which were associated with an increase of IFN-y-producing γ δ tumor infiltrating-lymphocytes (TILs) contributing also for anti-tumor CTX effect [126]. Therefore, cyclophospha-

Chemotherapy efficacy can also be impacted by intratumoral bacteria. Geller et al. (2017) showed, in a colon cancer mouse model, that Gammaproteobacteria can metabolize chemotherapeutic gemcitabine into an inactive form inducing chemotherapy resistance and that this effect was reversed by antibiotic ciprofloxacin. Interestingly, about 76% of human pancreatic ductal adenocarcinomas were positive for bacteria, mainly Gammaproteobacteria [127]. Perhaps the treatment for

Several studies have shown a complex crosstalk between bacteria and immune host response in the anti-tumor battle. For example, Paulos et al. (2007) reported

this tumor type may be improved by adding antibiotics to the chemotherapy. Chemotherapy-induced diarrhea (CD) is a frequent adverse event in dogs, in which changes in gut microbiota appear to play a key role. A recent study of 60 dogs undergoing chemotherapy supported the administration of smectite, a natural medical clay, widely used in acute diarrhea treatment in humans, as a first-line treatment of CD in dogs. Interestingly, smectite has anti-inflammatory properties to decrease intestinal bacterial translocation and stabilize intestinal microbiome [128]. However, studies associating microbiome and effectiveness of chemotherapy

mide immunomodulatory effects require a functional microbiome.

Some of the proposed mechanisms will be discussed below.

**4.1 Roles of microbiome in cancer therapy**

patients treated with chemotherapy.

are still scarce in veterinary medicine.

*4.1.2 Immunotherapy*

**124**

that total body irradiated mice showed a more efficient anti-tumor response to adoptively transferred tumor-specific CD8+ T cells against melanoma after gut microbial translocation to mesenteric lymph nodes. They observed that the radiation induced the release of microbial LPS and activated innate immune response by TLR4 stimulation and then increased anti-tumor CD8+ T cells, while reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling were associated with a decrease of anti-tumor response [129].

There is also evidence that gut microbiome modulates efficacy of immune checkpoint inhibitors (CIs), that are monoclonal antibodies with inhibitory effect to specific receptors on T cells and tumor cells, blocking signaling pathways that negatively modulate immune system, allowing specific T cells to promote destruction of cancer cells [130]. Those receptors include cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 protein (PD-1), and programmed death-ligand 1 (PD-L1) [131].

It was demonstrated that oral administration of Bifidobacterium in mice with melanoma was associated with the same degree of antitumor effects as in those mice that received therapy with PD-L1 antibodies, and the combination of both treatments almost abolished tumor growth [132]. In addition, a report of human gut microbiome metagenomic profiling in 39 metastatic melanoma patients treated with anti-PD1 and/or anti-CTLA-4 immunotherapy identified that those who respond to all types of CIs were enriched for *Bacteroides caccae*, enhancing that microbiota may modulate cancer immunotherapy [133]. These correlations have not yet been demonstrated in dogs.

## **4.2 Therapeutic manipulation of microbiome and its relevance to cancer therapies response**

Given the increasing evidence on the significant role that microbiome can play in cancer, microbiota modulation represents a new therapeutic potential capable of altering disease development. These therapies aim to change the microbial community associated with dysbiosis for those associated with health. In small animals, microbiome manipulations are often described as part of gastrointestinal diseases treatment [134]. Mainly as an adjuvant treatment for cancer, these interventions and their effectiveness are not well established and have only recently been described in literature. The following will discuss some ways in which microbiome can be modified:

## *4.2.1 Prebiotics and symbiotics*

Prebiotics are specific chemicals, capable of promoting growth of a selective group of bacteria and their specific metabolites and thus modulating microbiota in a beneficial way, which may help on anti-tumor treatment [135]. These are non-digestible or absorbable dietary fibers and include fructans (oligofructose and inulin), nonstarch polysaccharides found in some cereal grains, algae, disaccharides (lactulose), and polysaccharides including fructooligosaccharides (FOS) [22].

According to Villegér and colleagues, the effect of prebiotics depends on the presence of beneficial bacteria in the host's intestines [33]. Thus, the combination of probiotics and prebiotics, known as symbiotic, looks promising. Dietary treatment with inulin or oligofructose has been demonstrated to selectively stimulate growth of specific bacterial taxa and alter SCFA levels within the gut [136]. Moreover, these prebiotics reduced the incidence of mammary tumors in rats, significantly potentiated chemotherapy effects as well as RT [136]. The perioperative administration of symbiotics, probiotics (strains Lactobacillus and Bifidobacterium) and prebiotic

(fructooligosaccharides), reduced postoperative mortality and complication rates in cancer patients undergoing surgery [137].

The effects of prebiotics were evaluated in dogs, but without focusing on the benefits of cancer treatment. A recent study evaluated the effects of prebiotics in different concentrations in healthy adult dogs and concluded that the galactooligosaccharide prebiotic at 1.0% improved the immunity of healthy dogs [138]. Inulin intervention resulted in a modulation of intestinal bacteria, increase of fecal SCFA and BA in dogs. Given that some studies showed similar dysbiotic states between dogs and humans with cancer [12, 13, 37], it seems relevant that the new approaches to increase anticancer therapy efficiency should include the potential benefits of prebiotic supplementation for both dogs and humans.

#### *4.2.2 Probiotics*

Probiotics refers to live bacteria that can be orally administered and confer health beneficial when delivered in adequate amounts [139]. Probiotics colonize the gut temporarily and act modifying colonic environment. Different mechanisms are involved in probiotics protective role: increase in barrier function, epithelial tight junctions integrity, immune response modulation, anti-inflammatory cytokines production, pathogenic bacteria growth inhibition by antimicrobial and antitoxin compound production (i.e. SCFA), and production of enzymatic activities and/or beneficial metabolites to the host [140]. A recent systematic review and meta-analysis investigated probiotics efficacy and safety in patients diagnosed with cancer and concluded that probiotics may be beneficial but further studies are still required [141].

The strains of Lactobacillus and Bifidobacterium are most frequently reported in studies with probiotics. The "protective" effect against colorectal cancer was demonstrated after oral supplement containing *Lactobacillus helveticus* in mice with colonic cancer, in which tumor growth rate and degree of hyperplasia were reduced [142]. These effects were secondary to suppression of NF-κB, increased of anti-inflammatory IL-10 and decreased IL-17-producing T cells [142]. In addition, administration of *L. acidophilus* in mice with breast tumors reduced tumor growth due to altered cytokine production and, in a murine melanoma model, the therapy with aerosolized *L. rhamnosus* promoted immunity against lung metastases, identifying a role for a probiotic cancer "preventing" [143, 144].

Probiotics can also affect patient "outcomes". In a prospective randomized study, after transurethral resection of bladder cancer, the group of patients who received oral supplementation with *L. casei* associated with intravesical epirubicin application had a 3-year recurrence-free survival rate significantly higher than in the isolated chemotherapy group [145]. In addition, some studies demonstrate the action of probiotics on treatment-related toxicity. *L. rhamnosus* decreased diarrhea and abdominal discomfort in patients with colon cancer treated with 5-fluorouracil chemotherapy [146]. Symbiotics (a combination of *Bifidobacterium breve* and *L. casei*) during neoadjuvant chemotherapy in esophageal cancer patients reduced the occurrence of adverse events (diarrhea, neutropenia and lymphopenia) [147].

However, caution should be exercised in their use, since the composition of commercially available probiotics have been inadequately studied, as well as their long-term impact on intestinal microbiota and general health [135]. When investigating the use of pre- and probiotics in dogs, scientific evidence of their benefit is scarce, especially in cancer. Furthermore, the knowledge about appropriate doses and compositions is small in companion animals [148]. Studies suggest that they may have beneficial effects on canine IBD. In a prospective randomized study, 34 IBD dogs received prednisone with or without multi-strain probiotic. Both treatments increased the numbers of total bacteria and were associated with rapid

**127**

*Small Animals Gut Microbiome and Its Relationship with Cancer*

clinical remission but not improvement in histopathologic inflammation [149]. A protective effect of multi-strain probiotic (strains of Lactobacillus, Bifidobacterium and Streptococcus) was also observed in dogs with IBD compared with a control group (treated with metronidazole and prednisolone), with a significant decrease

A recent study showed that probiotics consumption (*L. casei*, *L. plantarum* and Bifidobacterium) in healthy dogs of different age groups, significantly increased beneficial intestinal bacteria (Lactobacillus and *Faecalibacterium prausnitzii*) and decreased potentially harmful bacteria (*E. coli* and *Sutterella stercoricanisin*) mostly in elderly dogs, suggesting that probiotic treatment improves host health and

In FMT, feces are transferred from a healthy donor to the intestinal tract of a diseased recipient [30]. FMT may be delivered via colonoscopy, enema or oral administration, with equal clinical efficacy [152]. The beneficial mechanisms of FMT are still unknown. Nowadays, FMT has been used in resistant *Clostridium difficile* treatment with high response rates [153]. Contrary to gastrointestinal (GI) diseases, application of FMT in cancer is still limited and data was obtained mainly in animal models. The reconstitution of germ-free mice with fecal material from patients with melanoma responsive to anti-PD-L1 and to anti-PD-1 therapies led to better tumor control in contrast to those that received faces from unresponsive

The use of FMT in veterinary medicine was studied mostly in dogs with GI diseases, such as in parvovirus-infected puppies and patients with diarrhea due to IBD and *C. perfringens*, and it was associated with faster resolution of clinical signs [156]. For a deep learning regarding the FMT effects in veterinary non-oncological diseases and the potential applications of FMT in animals, including therapeutic, prophylactic and immunogenic uses, the reader may consult Niederwerder (2018)

Host-microbiota interactions are crucial in human and animal health and disease development, yet microbiota function and dynamics during disease states are only partially understood. There is growing evidence supporting that immunoregulatory and anti-inflammatory effects of gut and tumor microbiota are essential in the battle of cancer. However, most studies were performed in preclinical models, which have many pitfalls in regard of spontaneous cancer research urging the need

This manuscript received no specific grant from any funding agency in the

*DOI: http://dx.doi.org/10.5772/intechopen.95780*

in clinical and histological scores [150].

*4.2.3 Fecal microbiome transplantation (FMT)*

immunity [151].

patients [154, 155].

publication [157].

**5. Conclusions**

**Acknowledgements**

**Conflict of interest**

for clinical studies benefiting both species.

public, commercial, or not-for-profit sectors.

The authors declare no conflict of interest.

*Small Animals Gut Microbiome and Its Relationship with Cancer DOI: http://dx.doi.org/10.5772/intechopen.95780*

*Canine Genetics, Health and Medicine*

*4.2.2 Probiotics*

cancer patients undergoing surgery [137].

prebiotic supplementation for both dogs and humans.

(fructooligosaccharides), reduced postoperative mortality and complication rates in

The effects of prebiotics were evaluated in dogs, but without focusing on the benefits of cancer treatment. A recent study evaluated the effects of prebiotics in different concentrations in healthy adult dogs and concluded that the galactooligosaccharide prebiotic at 1.0% improved the immunity of healthy dogs [138]. Inulin intervention resulted in a modulation of intestinal bacteria, increase of fecal SCFA and BA in dogs. Given that some studies showed similar dysbiotic states between dogs and humans with cancer [12, 13, 37], it seems relevant that the new approaches to increase anticancer therapy efficiency should include the potential benefits of

Probiotics refers to live bacteria that can be orally administered and confer health beneficial when delivered in adequate amounts [139]. Probiotics colonize the gut temporarily and act modifying colonic environment. Different mechanisms are involved in probiotics protective role: increase in barrier function, epithelial tight junctions integrity, immune response modulation, anti-inflammatory cytokines production, pathogenic bacteria growth inhibition by antimicrobial and antitoxin compound production (i.e. SCFA), and production of enzymatic activities and/or beneficial metabolites to the host [140]. A recent systematic review and meta-analysis investigated probiotics efficacy and safety in patients diagnosed with cancer and concluded

The strains of Lactobacillus and Bifidobacterium are most frequently reported in studies with probiotics. The "protective" effect against colorectal cancer was demonstrated after oral supplement containing *Lactobacillus helveticus* in mice with colonic cancer, in which tumor growth rate and degree of hyperplasia were reduced [142]. These effects were secondary to suppression of NF-κB, increased of anti-inflammatory IL-10 and decreased IL-17-producing T cells [142]. In addition, administration of *L. acidophilus* in mice with breast tumors reduced tumor growth due to altered cytokine production and, in a murine melanoma model, the therapy with aerosolized *L. rhamnosus* promoted immunity against lung metastases,

Probiotics can also affect patient "outcomes". In a prospective randomized study, after transurethral resection of bladder cancer, the group of patients who received oral supplementation with *L. casei* associated with intravesical epirubicin application had a 3-year recurrence-free survival rate significantly higher than in the isolated chemotherapy group [145]. In addition, some studies demonstrate the action of probiotics on treatment-related toxicity. *L. rhamnosus* decreased diarrhea and abdominal discomfort in patients with colon cancer treated with 5-fluorouracil chemotherapy [146]. Symbiotics (a combination of *Bifidobacterium breve* and *L. casei*) during neoadjuvant chemotherapy in esophageal cancer patients reduced the occurrence of adverse events (diarrhea, neutropenia and lymphopenia) [147]. However, caution should be exercised in their use, since the composition of commercially available probiotics have been inadequately studied, as well as their long-term impact on intestinal microbiota and general health [135]. When investigating the use of pre- and probiotics in dogs, scientific evidence of their benefit is scarce, especially in cancer. Furthermore, the knowledge about appropriate doses and compositions is small in companion animals [148]. Studies suggest that they may have beneficial effects on canine IBD. In a prospective randomized study, 34 IBD dogs received prednisone with or without multi-strain probiotic. Both treatments increased the numbers of total bacteria and were associated with rapid

that probiotics may be beneficial but further studies are still required [141].

identifying a role for a probiotic cancer "preventing" [143, 144].

**126**

clinical remission but not improvement in histopathologic inflammation [149]. A protective effect of multi-strain probiotic (strains of Lactobacillus, Bifidobacterium and Streptococcus) was also observed in dogs with IBD compared with a control group (treated with metronidazole and prednisolone), with a significant decrease in clinical and histological scores [150].

A recent study showed that probiotics consumption (*L. casei*, *L. plantarum* and Bifidobacterium) in healthy dogs of different age groups, significantly increased beneficial intestinal bacteria (Lactobacillus and *Faecalibacterium prausnitzii*) and decreased potentially harmful bacteria (*E. coli* and *Sutterella stercoricanisin*) mostly in elderly dogs, suggesting that probiotic treatment improves host health and immunity [151].

#### *4.2.3 Fecal microbiome transplantation (FMT)*

In FMT, feces are transferred from a healthy donor to the intestinal tract of a diseased recipient [30]. FMT may be delivered via colonoscopy, enema or oral administration, with equal clinical efficacy [152]. The beneficial mechanisms of FMT are still unknown. Nowadays, FMT has been used in resistant *Clostridium difficile* treatment with high response rates [153]. Contrary to gastrointestinal (GI) diseases, application of FMT in cancer is still limited and data was obtained mainly in animal models. The reconstitution of germ-free mice with fecal material from patients with melanoma responsive to anti-PD-L1 and to anti-PD-1 therapies led to better tumor control in contrast to those that received faces from unresponsive patients [154, 155].

The use of FMT in veterinary medicine was studied mostly in dogs with GI diseases, such as in parvovirus-infected puppies and patients with diarrhea due to IBD and *C. perfringens*, and it was associated with faster resolution of clinical signs [156]. For a deep learning regarding the FMT effects in veterinary non-oncological diseases and the potential applications of FMT in animals, including therapeutic, prophylactic and immunogenic uses, the reader may consult Niederwerder (2018) publication [157].

#### **5. Conclusions**

Host-microbiota interactions are crucial in human and animal health and disease development, yet microbiota function and dynamics during disease states are only partially understood. There is growing evidence supporting that immunoregulatory and anti-inflammatory effects of gut and tumor microbiota are essential in the battle of cancer. However, most studies were performed in preclinical models, which have many pitfalls in regard of spontaneous cancer research urging the need for clinical studies benefiting both species.

#### **Acknowledgements**

This manuscript received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Canine Genetics, Health and Medicine*
