Canine Disease Research Articles

Genomic Medicine in Canine Periodontal Disease: A Systematic Review.

Genomic medicine has become a growing reality; however, it is still taking its first steps in veterinary medicine. Through this approach, it will be possible to trace the genetic profile of a given individual and thus know their susceptibility to certain diseases, namely periodontal disease. This condition is one of the most frequently diagnosed in companion animal clinics, especially in dogs. Due to the limited existing information and the lack of comprehensive studies, the objective of the present study was to systematically review the existing scientific literature regarding genomic medicine in canine periodontal disease and determine which genes have already been studied and their probable potential. This study followed the recommendations of the PRISMA 2020 methodology. Canine periodontal disease allied to genomic medicine were the subjects of this systematic review. Only six articles met all of the inclusion criteria, and these were analyzed in detail. These studies described genetic variations in the following genes: interleukin-6, interleukin-10, interleukin-1, lactotransferrin, toll-like receptor 9, and receptor activator of nuclear factor-kappa B. Only in two of them, namely interleukin-1 and toll-like receptor 9 genes, may the identified genetic variations explain the susceptibility that certain individuals have to the development of periodontal disease. It is necessary to expand the studies on the existing polymorphic variations in genes and their relationship with the development of periodontal disease. Only then will it be possible to fully understand the biological mechanisms that are involved in this disease and that determine the susceptibility to its development.

Clinical assessment of a point-of-care assay to determine protective vaccinal antibody titers to canine viral diseases

Guidelines recommend that dogs are vaccinated for canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) every 3 years. Alternatively, their antibody titers are measured and vaccines given when titers fall below a protective threshold. In this study, a point-of-care (POC) assay was compared to hemagglutination inhibition (for CPV) and virus neutralization (for CAV and CDV) assays to predict the need for revaccination Ninety-two dogs presented for vaccination were enrolled. The POC assay indicated protective titers against CDV in 79/80, CPV in 89/90, and CAV in 91/91 dogs with reference standard antibody measurements that were over a protective threshold. The sensitivity of the POC assay for to detect protective concentrations of CDV antibodies was 99% (95% confidence interval [CI 95%], 93.3–99.9%). Ten dogs were falsely considered protected against CDV by the POC assay with a specificity of 17% (CI 95%, 3.0–44.8%). The sensitivity of the POC assay for protective concentrations of CPV titers was 99% (CI 95%, 93.9–99.9%). The sensitivity of the POC assay to detect protective concentrations of CAV antibodies was 100% (CI 95%, 95.9–100%). Only classifying high-positive CDV and CPV titers on the POC assay as protective improved assay specificity to 100%, but sensitivity decreased to 51% and 76% respectively. This POC assay had a high sensitivity for the detection of protective antibody titers; however, some dogs were falsely categorized as protected, especially for CDV.

Droplet digital PCR as a tool to detect resistant isolates of Dirofilaria immitis

Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.

Efficacy of an oral combination of moxidectin, afoxolaner, and pyrantel pamoate for the prevention of heartworm disease in dogs

Dirofilaria immitis, the mosquito-borne agent of dirofilariosis, a chronic and sometimes fatal cardiopulmonary canine disease, is endemic in most warm and temperate regions in the world. The efficacy of an oral endectoparasiticide product (test product or TP) combining moxidectin, afoxolaner, and pyrantel pamoate was evaluated for the prevention of heartworm disease in dogs, in two laboratory and one field studies. In each laboratory study, 20 D. immitis-naïve beagle dogs were experimentally infected with D. immitis. Ten control dogs were sham-treated, and ten dogs were administered the TP targeting the minimum effective dose, six times monthly and starting 30 days post infection. At necropsy seven months after inoculations, no heartworms were found in any of the TP treated dog, whereas 19 to 42 live heartworms were found in the control dogs. In each study, treatment efficacy was 100% and the difference between treated and untreated groups was highly significant (p < 0.0001). A field study was conducted through the full transmission season in several heartworm-endemic regions of the United States. One hundred and twenty client-owned dogs that were negative for D. immitis at enrollment were administered twelve monthly oral doses of the TP at label dose. Blood tests for D. immitis antigen and modified Knott's tests for microfilariae remained negative through the full duration of the study, demonstrating that all dogs were protected from heartworm infection during the full transmission season. These studies demonstrated that TP administered monthly for at least six doses is effective at preventing dirofilariosis.

Stakeholder opinion-led study to identify canine priority diseases for surveillance and control in the UK.

Many pathogens cause disease in dogs; however, meaningful surveillance in small companion animals is often only possible for the most impactful diseases. We describe the first stakeholder opinion-led approach to identify which canine infectious diseases should be prioritised for inclusion in surveillance and control strategies in the UK. Participants were identified through a stakeholder analysis. A multicriteria decision analysis was undertaken to establish and weight epidemiological criteria for evaluating diseases, and a Delphi technique was employed to achieve a consensus among participants on the top-priority canine diseases. Nineteen stakeholders from multiple backgrounds participated in this study. Leptospirosis and parvovirus were identified as the top two endemic diseases of concern, while leishmaniosis and babesiosis were the top two exotic diseases. Respiratory and gastrointestinal diseases were identified as the top two syndromes of concern. Due to the COVID-19 pandemic, the number of participants was reduced. Despite this, a representative multidisciplinary sample of relevant stakeholders contributed to the present study. Findings from this study are being used to inform the development of a future UK-wide epidemic response strategy. This methodology could provide a blueprint for other countries.

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Dirofilaria immitis Identified in Galapagos Sea Lions (Zalophus wollebaeki): A Wildlife Health and Conservation Concern.

The Galapagos sea lion (Zalophus wollebaeki), an endemic and endangered pinniped, faces an increasing threat due to infectious diseases related to domestic animals. Dirofilaria immitis, the parasite responsible for canine heartworm disease, is one such threat, as canine infections on the archipelago have been documented. We used a canine heartworm antigen test kit to analyze the blood from 25 juvenile Galapagos sea lions for D. immitis. Two (8%) sea lions tested positive for D. immitis antigen. Using morphologic and genetic assessments, we evaluated 20 filarial-like worms collected from within the heart of an adult male Galapagos sea lion during a previous routine postmortem examination. The intracardiac worms were morphologically consistent with adult D. immitis, and sequence analysis of targeted PCR amplicons confirmed their identity. This is the first report of D. immitis infection in Galapagos sea lions, which could become a major health problem for these pinnipeds. Further studies are necessary to confirm the level of threat from this parasite; however, widespread adoption of routine heartworm testing, prevention, and treatment in the canine population, and the control of mosquitos, could potentially reduce the disease impact on this endangered pinniped species.

MiRNAs: Potential as Biomarkers and Therapeutic Targets for Cancer.

MicroRNAs (miRNAs) are single-stranded, non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to messenger RNAs. miRNAs are important regulators of gene expression, and their dysregulation is implicated in many human and canine diseases. Most cancers tested to date have been shown to express altered miRNA levels, which indicates their potential importance in the oncogenic process. Based on this evidence, numerous miRNAs have been suggested as potential cancer biomarkers for both diagnosis and prognosis. miRNA-based therapies have also been tested in different cancers and have provided measurable clinical benefits to patients. In addition, understanding miRNA biogenesis and regulatory mechanisms in cancer can provide important knowledge about resistance to chemotherapies, leading to more personalized cancer treatment. In this review, we comprehensively summarized the importance of miRNA in human and canine cancer research. We discussed the current state of development and potential for the miRNA as both a diagnostic marker and a therapeutic target.

Randomized single-blinded prospective comparison between ozone therapy and electroacupuncture for canine thoracolumbar disk disease

Ozone therapy has been used to control pain in animals. Moreover, electroacupuncture (EA) has been effective in neurological recovery and pain control in dogs with thoracolumbar discopathy. EA was compared with Ozone applied at acupuncture points in dogs with signs of thoracolumbar disk disease. Chondrodystrophic mongrel dogs, with lesion scores between 1 and 4, were randomly divided into groups: EA (n = 13) - electroacupuncture at BL20, BL23, ST36, KID3 and BL60 and dry needle in lumbar Bai Hui, and OZO (n = 15) - paravertebral application of 3 mL ozone (20 μg / mL) at BL20, BL23, lumbar Bai Hui, ST36, and KID3/BL60, both groups with weekly applications. No significant differences were observed between groups in weekly blind pain assessments using a dynamic interactive visual analog scale, and neurological assessments using a numerical-functional scale. Both groups showed a progressive improvement in pain control and neurological condition, which was observed when comparing the group's EA and OZO in dogs with all lesion scores. The time of return to locomotion (days) of dogs scored 3 and 4, between groups EA (10.6 ± 5.4) and OZO (14.5 ± 15.7), had no significant differences. Ozone therapy was effective to control pain and motor rehabilitation and sensory functions of dogs with thoracolumbar discopathy signs, just as electroacupuncture. Ozone application was a quick and easy-to-handle treatment. Paravertebral and subcutaneous routes did not require anesthesia or other advanced imaging technology, besides being safe and effective.

Seroprevalence of canine respiratory coronavirus in breeding kennels in Croatia

Canine respiratory coronavirus is a relatively new addition to the list of pathogens causing canine infectious disease complex. The virus is highly contagious, with a high prevalence in the dog population worldwide, especially in shelters. This study aimed to establish the presence and risk factors associated with infection in privately owned dogs and breeding colonies. This study was the first to demonstrate the presence of canine respiratory coronavirus in Croatia. Out of the 257 serum samples, 35.03% of dogs from breeding kennels and 43% of pet dogs tested enzyme-linked immunoassay positive, but the difference was not statistically significant. Sex was not an important risk factor, but the seropositivity rate increased with age. Mixing of dogs during hunting, training and dog shows was not associated with a higher seroprevalence in the breeding colonies. Daily cleaning and disinfection showed little effect on the infection spread. The study was done on a limited sample. However, it still provides evidence that the epizootiology of this disease is complex. There is no available vaccine for canine respiratory coronavirus, and further studies on environmental and risk factors will give the valuable data needed to prevent this disease.

Canine gastrointestinal parasites perceptions, practices, and behaviours: A survey of dog owners in Australia

Many species of canine gastrointestinal (GI) parasites are known to be zoonotic meaning that dog owners' management and practices are key to preventing exposure of humans and dogs as well as contamination of the environment. As Australia has one of the highest rates of pet ownership in the world, we administered an online questionnaire to dog owners across the nation to assess their perceptions, practices, and behaviours towards canine GI parasites. Descriptive analysis was performed to summarise perceptions and management practices. Factors associated with the suitability of parasiticide treatments applied were investigated using uni- and multivariable ordinal regression. Just over a half of dog owners considered parasites as very or extremely important for their dog's health (59%) and less than a half as very or extremely important for human health (46%). Although the majority of dog owners stated that they deworm their dogs (90%), only the 28% followed best practice guidelines, i.e. administered a monthly prophylactic treatment all-year round. A large proportion of respondent dog owners administered prophylactic treatment at an inappropriate frequency (48%) or did not treat for canine GI parasites at all (24%).Attending vet visits at least once a year or once every six months and having a very comfortable or prosperous financial position were significantly associated with following best deworming prophylaxis guidelines. This study demonstrates that a proportion of dog owners in Australia is not complying with best practice regarding the control of canine GI parasites and is potentially exposing themselves and their dogs to the risk of infections. Veterinarians are called to implement dog owner's education, raise their awareness on the threats canine parasitic diseases pose to both dogs and humans and finally, encourage them to follow a monthly prophylactic treatment for canine GI parasites all year round.

Characterization of canine intestinal microRNA expression in inflammatory bowel disease and T-cell lymphoma

Differentiating between canine inflammatory bowel disease (IBD) and intestinal T-cell lymphoma by histopathological examination of endoscopically-derived intestinal biopsies can be challenging and involves an invasive procedure requiring specialized equipment and training. A rapid, non-invasive method of diagnosis, such as blood or faecal analysis for a conserved and stable biomarker, would be a useful adjunct or replacement. Studies on dogs and humans with various types of lymphoma have shown altered microRNA (miRNA) expression patterns in blood, faeces and tissues indicating their potential use as biomarkers of disease. The present study used residual archived endoscopically-derived, formalin-fixed, paraffin-embedded (FFPE) duodenal tissue taken from pet dogs undergoing routine investigation of gastrointestinal disease. The dogs had previously been diagnosed with either normal/minimal intestinal inflammation, severe IBD or intestinal T-cell lymphoma. Next generation sequencing with qPCR validation was used to elucidate differentially expressed miRNAs between groups. Our results show that miRNA can be extracted from archived endoscopically-derived FFPE tissues from the canine duodenum and used to differentiate normal/minimally inflamed canine duodenal tissue from severe lymphoplasmacytic IBD and T-cell lymphoma.

Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Predicting health outcomes in dogs using insurance claims data

In this paper we propose a machine learning-based approach to predict a multitude of insurance claim categories related to canine diseases. We introduce several machine learning approaches that are evaluated on a pet insurance dataset consisting of 785,565 dogs from the US and Canada whose insurance claims have been recorded over 17 years. 270,203 dogs with a long insurance tenure were used to train a model while the inference is applicable to all dogs in the dataset. Through this analysis we demonstrate that with this richness of data, supported by the right feature engineering, and machine learning approaches, 45 disease categories can be predicted with high accuracy.

Molecular Survey and Genetic Characteristics of Vector-Borne Pathogens in Domestic Dogs from Four Regions of China.

Canine vector-borne diseases are widely distributed around the world. They are transmitted by arthropods, and many seriously threaten the health of animals and humans. In China, our knowledge of Ehrlichia, Hepatozoon, and Mycoplasma species circulating in dogs is still poorly understood. Therefore, the aim of this study was to understand the prevalence and genetic characteristics of canine Ehrlichia spp., Hepatozoon spp., and Mycoplasma spp. in Chongqing (southwest), Fujian (southeast), Shandong (southeast), and Hubei (central) Provinces of China. Blood samples from healthy pet dogs were processed to detect Ehrlichia, Hepatozoon, and Mycoplasma DNA with PCR. Haplotype and phylogenetic analyses were performed on 18S rRNA sequences. Among 306 dogs, no Ehrlichia spp. or Mycoplasma spp. were detected, whereas one Hepatozoon sp. was detected in 10 (3.27%) of the animals. Only Hepatozoon canis was identified and was endemic to Chongqing (2.46%) and Hubei (8.77%). A haplotype analysis identified eight haplotypes among the H. canis isolates. A phylogenetic analysis showed that the H. canis isolates in this study clustered into four clades, together with isolates from different countries and hosts, forming a large group that was clearly separate from other Hepatozoon species. These findings provided new information on the epidemiological characteristics of canine vector-borne diseases in China and will be helpful in the development of efficient measures to safeguard the health and well-being of companion animals and their owners.

In canine cranial cruciate ligament disease, are conservative treatment and lateral fabellotibial suture recoveries comparable?

PICO question In dogs with cranial cruciate ligament disease, is conservative non-surgical treatment as effective as surgical treatment with the lateral fabellotibial suture technique in reducing time to recovery? Clinical bottom line Category of research Treatment. Number and type of study designs reviewed One retrospective study. Strength of evidence Zero. Outcomes reported There is no evidence that surgical treatment with the lateral fabellotibial suture (LFTS) reduces time to recovery compared to conservative treatment in dogs with cranial cruciate ligament disease. Conclusion In dogs with cranial cruciate ligament disease there is no statistical evidence to support recommendation of surgical treatment with the LFTS technique compared to conservative treatment in reducing time to recovery. How to apply this evidence in practice The application of evidence into practice should take into account multiple factors, not limited to: individual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where you work, the individual case in front of you, the availability of therapies and resources. Knowledge Summaries are a resource to help reinforce or inform decision making. They do not override the responsibility or judgement of the practitioner to do what is best for the animal in their care.

Faecal microbiome transplantation improves clinical signs of chronic idiopathic large bowel diarrhoea in working dogs.

Chronic diarrhoea is a common clinical sign in dogs with chronic enteropathy, and psyllium husk has been shown to improve clinical signs in affected dogs. The aim of this study was to investigate whether faecal microbiome transplant has a similar effect in alleviating clinical signs in dogs with chronic large bowel diarrhoea. Thirty large-breed working dogs with chronic large bowel diarrhoea were divided into a psyllium group (PG) and a faecal microbiome transplant group (FMTG). To the PG, 16g/day of psyllium husk was administered for 30 days. The FMTG received faecal microbiome transplantation (FMT) once via enema. A daily log of faecal characteristics was kept, and the dogs' canine inflammatory bowel disease index (CIBDAI) and body condition scores (BCS) were determined. A Wilcoxon-Mann-Whitney test was used to compare group results. In addition, the Kaplan-Meier test was used to evaluate the occurrence rate of 1 day or more of diarrhoea and 2 days or more of diarrhoea by day 30. The sample had a mean age of 3.9±2.1 years and a bodyweight of 25.3±6.8kg. The FMTG showed a more rapid onset of CIBDAI improvement but no difference in other measures. At 30 days, the FMTG showed a greater improvement in bodyweight and BCS, but no differences were observed in faecal scores, defaecation frequency and time of appearance of episodes of diarrhoea. Time played a significant positive role in the results observed across both groups (p < 0.05). This study did not compare the microbiomes of the dogs before and after treatment, so the role of specific types of bacteria cannot be determined. Psyllium husk and FMT had similar effects in improving clinical signs of chronic large bowel diarrhoea.

Mucosal Vaccination with Live Attenuated Bordetella bronchiseptica Protects against Challenge in Wistar Rats.

Bordetella bronchiseptica (Bb) is a Gram-negative bacterium responsible for canine infectious respiratory disease complex (CIRDC). Several vaccines targeting this pathogen are currently licensed for use in dogs, but their mechanism of action and the correlates of protection are not fully understood. To investigate this, we used a rat model to examine the immune responses induced and the protection conferred by a canine mucosal vaccine after challenge. Wistar rats were vaccinated orally or intranasally on D0 and D21 with a live attenuated Bb vaccine strain. At D35, the rats of all groups were inoculated with 103 CFU of a pathogenic strain of B. bronchiseptica. Animals vaccinated via either the intranasal or the oral route had Bb-specific IgG and IgM in their serum and Bb-specific IgA in nasal lavages. Bacterial load in the trachea, lung, and nasal lavages was lower in vaccinated animals than in non-vaccinated control animals. Interestingly, coughing improved in the group vaccinated intranasally, but not in the orally vaccinated or control group. These results suggest that mucosal vaccination can induce mucosal immune responses and provide protection against a Bb challenge. This study also highlights the advantages of a rat model as a tool for studying candidate vaccines and routes of administration for dogs.

Congenital ventricular outflow tract obstructions in Boxer dogs: Results of a 17-year cardiovascular breed screening program in France (3126 dogs).

Ventricular outflow tract obstructions including aortic (AS) and pulmonic stenosis (PS) are the most common canine congenital heart diseases, with Boxer dogs being predominantly affected. This has led to the French Boxer club adopting a mandatory national control program against AS and PS. The objective of this retrospective study was to analyze the results of 17 years of this cardiovascular breed screening program (2005-2021). The records of untreated and non-anesthetized adult Boxer dogs screened between 2005 and 2021 were retrospectively reviewed. All dogs underwent physical examination and standard transthoracic echocardiography with concomitant ECG tracing. All examinations were reviewed by one single board-certified specialist in cardiology. Out of the 3126 dogs screened during the study period, 3001 dogs (female:male sex ratio = 2.2, median age [IQR] = 1.6 years [1.2-2.1]) were recruited for data analysis. A total of 218 operators were involved in the screening program. For most Boxer dogs (i.e., 93.8% for AS and 94.5% for PS), a single examination was required to obtain a definitive cardiac status, although most operators were non-specialist general practitioners. A left basilar systolic heart murmur was detected in all dogs with AS and PS, but also in 7.4% dogs free of heart diseases. A significantly higher proportion of the latter was detected when operators were board-certified specialists (P<0.001). Lastly, when comparing the start and the end of the breeding program, among dogs diagnosed with AS and PS (n = 364) in a French referral cardiology center, Boxer went from the 1st affected breed by AS to the 3rd, and from the 3rd affected breed by PS to the 6th. This 17-year screening program has experienced a strong involvement of veterinarians, breeders, and owners throughout France. This may have contributed to reduce AS and PS prevalence in Boxer dogs at the studied referral cardiology center.

Use of In-Clinic Diagnostic Kits for the Detection of Seropositivity to Leishmania infantum and Other Major Vector-Borne Pathogens in Healthy Dogs.

Canine Vector-Borne Diseases (CVBDs) are widespread in Europe and enzootic in many other countries. Though severe illnesses may occur, dogs living in enzootic areas often show vague or no clinical signs of CVBDs. Undiagnosed infections/co-infections in subclinically infected animals favor the spread of CVBDs and increase the risk of transmission to other animals and, in some cases, humans. This study has evaluated the exposure of dogs living in key enzootic countries, i.e., Italy and Greece, to major CVBDs via the use of in-clinic diagnostic kits. Overall, 300 privately owned dogs without/with single mild clinical signs living in different regions of Italy (n. 150) and Greece (n. 150) were included in the study. As part of a clinical examination, a blood sample was collected from each dog and subjected to two serological rapid tests, i.e., the SNAP® 4Dx®Plus (IDEXX Laboratories Inc.) for the detection of antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi s.l. and Dirofilaria immitis antigen and the SNAP®Leishmania (IDEXX Laboratories Inc.) for the detection of antibodies against Leishmania infantum. In all, 51 dogs (17%; 95% CI 12.9-21.7) were seropositive to at least 1 pathogen, i.e., 4 in Italy (2.7%; 95% CI 1.4-13.1) and 47 in Greece (31.3%; 95% CI 24-39.4). Dirofilaria immitis antigens were found in 39 dogs (13%; 95% CI 9.4-17.3), while antibodies against Ehrlichia, Anaplasma and Leishmania were detected in 25 (8.3%; 95% CI 5.5-12.1), 8 (2.7%; 95% CI 1.2-5.2) and 5 (1.7%; 95% CI 0.5-3.8) dogs, respectively. None of the dogs tested seropositive for B. burgdorferi s.l. Statistical analyses were performed to evaluate associations between exposure to CVBDs and possible risk factors. The present results indicate that dogs living in enzootic areas may be seropositive for one or more CVBDs in absence of clinical signs. Rapid kits are among first line tools for the detection of CVBDs in clinical settings, as they are cost-effective, straightforward and quick to use. Also, in-clinic tests used herein allowed detection of co-exposure to CVBDs investigated.