Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott’s technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19–3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1–14.0; Caserta, OR=5.1, 95%CI: 2.5–10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6–4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1–3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (−0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

Acanthocheilonema reconditum is commonly considered an apathogenic parasite, as most studies do not describe clinical disease associated with confirmed infection Brianti et al. 2012;Otranto et al. 2013). In addition, Papazahariadou et al. (1994) reported no correlation between episodic weakness in hunting dogs and A. reconditum infection. In contrast, a purulent mesenteric lymphadenitis was reported by  as acute response in an experimentally infected dog. Martins Pereira et al. (2004) also described two erratic migrations and ectopic localizations of adult worms, with one dog having adult parasites in the heart (left ventricle), in association with bronchopneumonia and microfilariae in the alveoli, and the other with adult worms in the ovarian large ligament, occasionally found during ovariectomy. Other sporadic clinical abnormalities related with A. reconditum infection included subcutaneous suppurative inflammatory nodules in a naturally infected dog (Engelmann et al. 2019). Regarding clinical pathology, few reports have considered the occurrence of haematological and biochemical abnormalities in A. reconditum-infected dogs. A peripheral eosinophilia was found in experimentally infected dogs in the acute/invasive phase, while lymphocytosis developed during the chronic phase of infection ). In one study involving naturally infected dogs, Hashem and Badawy (2007) reported regenerative hypochromic anaemia, leucocytosis with neutrophilia, eosinophilia and monocytosis, increased serum liver enzymes (aspartate aminotransferase-AST, alanine aminotransferase-ALT and total bilirubin), and renal function parameters (blood urea nitrogen-BUN, creatinine, and serum inorganic phosphate).
Hunting dogs are frequently exposed to vector-borne pathogens due to their lifestyle, which is characterized by closer contact with wooded areas, cohabitation in outdoor kennels, and potentially less consistent use of antiparasitic drugs with limited care provided by owners (Piantedosi et al. 2017;Orr et al. 2020). Moreover, intense physical activity makes hunting dogs more susceptible to episodic weakness, as often reported by hunters. Diagnostic evaluation of episodic weakness can be challenging, and a previous study associated the presence of filariasis with exercise intolerance (Papazahariadou et al. 1994). While the pathogenesis of D. immitis microfilariae is well known, minimal data regarding the pathogenic potential role of A. reconditum microfilariae are available (McCall et al. 2008;Muñoz-Caro et al. 2018).
Based on the above premises, the aims of the present study were (i) to illustrate the distribution and the epidemiology of A. reconditum infection in dogs in Europe, (ii) to assess the prevalence and risk factors of A. reconditum infection in a large and specific hunting dog population from southern Italy, and (iii) to determine the frequency of haematological and serum biochemical abnormalities associated to A. reconditum infection as related to the microfilariae load.

Overview of Acanthocheilonema reconditum epidemiology in dogs in Europe
Acanthocheilonema reconditum (syn. Dipetalonema) was first described by Grassi in 1889. Grassi and Calandruccio (1890) defined morphological differences with D. immitis, by reporting details on an immature female worm; the same authors proved that A. reconditum develops in fleas. Additional data was reported by Noë (1907), who described further morphological features, analyzing some adult specimens (male and female) of A. reconditum. Additional morphological characteristic of A. reconditum microfilariae was subsequently reported by Newton and Wright (1956). Subsequent studies further defined the life cycle and completed the description of the parasite's morphology (Newton and Wright 1956;Nelson 1962;Bain and Beaucournu 1974;Korkejian and Edeson 1978). Acanthocheilonema reconditum infections are reported in several European countries, and this worm is considered the main filaroid species infecting dogs in the Mediterranean basin . Otranto et al. (2013) reported the geographical distribution of A. reconditum, showing its presence in Italy, Spain, Germany, Austria, and Greece. In another recent review, Tahir et al. (2019) described Acanthocheilonema infections (A. reconditum and A. dracunculoides), reporting the distribution in dogs and foxes in the Mediterranean basin, including the Middle East and northern Africa.
Full information about the prevalence of this parasite in dogs in Europe is scarce; most often its occurrence is a secondary finding within epidemiological surveys focused on other major Filaridae, such as D. immitis or D. repens.
In Italy, the presence of A. reconditum has been confirmed by different surveys in southern areas of the country (mean prevalence 7.7 %) (Cringoli et al. 2001;Giannetto et al. 2007;Brianti et al. 2012). In northern and central regions of Italy, a lower prevalence was reported (mean prevalence 1 %) Magi et al. 2012;Gizzarelli et al. 2019;Macchioni et al. 2020), but in contrast, a higher infection rate (7.7 %) was described in Liguria region by Magi et al. (2016). Two recent countrywide Italian surveys reported an overall A. reconditum prevalence of 0.8% and 0.1% (Brianti 2018¸Traversa et al. 2019. Among the other Mediterranean basin countries, presence of A. reconditum has been described in Greece, Cyprus, Turkey, Spain, Portugal, and France. In Greece, most studies are from the northern area of the country with a mean prevalence of 2.4 % (Papazahariadou et al. 1994;Founta et al. 1999;Diakou et al. 2016). In Cyprus, A. reconditum was the most represented among microfilaria species (Attipa et al. 2019;Kokkinos et al. 2019). In Turkey, Toparlak et al. (2005) showed A. reconditum as the only microfilaria species with a low prevalence (0.6 %) compared to the other Mediterranean countries. In Spain, a mean prevalence of 1.3 % was reported (Perez-Sanchez et al. 1989;Ortega-Mora et al. 1991;Aranda et al. 1998), with hotspots in a central-northern area of the country (Soria province-15.8%) and in the Canary Islands (20%) (Ortega-Mora et al. 1991) In the Iberian Peninsula, Portugal showed a lower prevalence, with a mean value of 0.55 % (Menn et al. 2010;Maia et al. 2016;Ferreira et al. 2017).
In France, the presence of A. reconditum was reported in different vector and life cycle studies, which contributed to the definitive classification of filarial species (Bain and Beaucournu 1974;Chauve 1990;Chabaud and Bain 1994). A very low prevalence (0.05%) was reported by Ducos de Lahitte in 1990. Despite several studies about filariosis, to the best of our knowledge, there are no recently published data on the prevalence of A. reconditum in France, except for a 2019 survey reporting negative results for A. reconditum infection (Laidoudi et al. 2019).
Regarding countries of central Europe, in Switzerland, Belgium, and the Netherlands, despite the ascertained presence of other filarial species, A. reconditum seems never to have been reported (Meyer et al. 1994;Deplazes et al. 1995;Bucklar et al. 1998;Petruschke et al. 2001;Overgaauw and van Dijk 2009;De Bosschere and Kindermans 2019).
Several studies involving A. reconditum have been carried out in Germany with most of the infections described in imported dogs (Zahler et al. 1997;Pantchev et al. 2011;Hamel et al. 2012;Schäfer et al. 2019), but only few autochthonous cases were reported in central Germany (0.19 %) (Liesner et al. 2016).
In the UK, there is only one dated study describing A. reconditum microfilariae in imported dogs from Ireland (Jacobs and Prole 1976).
In the Balkanian area, the presence of A. reconditum was described in the northern region of Serbia (2.1 %) (Tasić et al. 2008), although two subsequent studies in the same country found negative results (Tasić et al. 2012;Potkonjak et al. 2020). In Slovenia, Croatia, and Bulgaria, A. reconditum microfilaraemia was not detected in surveys on filariosis in dogs (Kirkova et al. 2007;Georgieva et al. 2001;Morchón et al. 2012;Holler et al. 2010;Radev et al. 2016).
In the eastern European countries, A. reconditum was reported in Romania (2.05 %) (Ionică et al. 2015), while in Slovakia, the parasite was not found (Miterpáková et al. 2010;Víchová et al. 2014). In Poland, A. reconditum was reported in 2011, when two dogs were found positive during a survey on other filarial species (Masny et al. 2011).

Study area
The study area included six different hunting districts (ATCs) in Campania and Basilicata regions of southern Italy. These are located in the provinces of Napoli (40°50′ N-14°15′ E) 7′ 00″ E) (ATC SA1), and Potenza (40°38′ 19″ N-15°48′ 08″ E) (ATC PZ). The territory of the Napoli, Caserta, Salerno, and Potenza provinces overlooks the Tyrrhenian Sea. The study region has a typical Mediterranean temperate climate along the coast, which becomes progressively continental in the inland and mountainous territories.

Study animals and sample size
The survey included 3020 hunting dogs and was performed in 81 private veterinary clinics located in the study area from 2014 to 2019; all procedures were performed for diagnostic purposes during a routine health check, and for this reason, the study did not require an ethical approval according to European Directive 2010/63/EU. Written informed consent was obtained from the owners of the hunting dogs included in the study.
The study was performed within a hunting dog's health assistance program of University of Naples, supported by the Italian management committees of the respective hunting districts (ATCs). In order to rule out the spread of major canine vector-borne pathogens in the study area, serological inclinic assay test systems [SNAP®4Dx®Plus (Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi s.l., D. immitis)], based on enzyme immunoassay technique, were employed on each  Kirkova et al. 2007 dog enrolled. Additionally, A. reconditum microfilaremic dogs were tested for antibodies to Leishmania infantum and for the presence of Angiostrongylus vasorum antigen, using the SNAP® Leishmania and IDEXX Angio Detect® Test kits, respectively, in order to rule out a possible coinfection with these two pathogens.
To better define A. reconditum infection risk, a questionnaire was administered to each owner to collect information about the dog's characteristics, habits, and management/ handling that might influence exposure and/or susceptibility to the parasite (see "Statistical analysis" for details). Because hunters from the Campania region have the habit of extralabel use of subcutaneous ivermectin injection for the prevention of ecto-and endoparasite infections (Piantedosi et al. 2017), owners were asked specifically in the questionnaire about macrocyclic lactone treatment schedule.
The necessary sample size to estimate prevalence of A. reconditum was calculated using the formula proposed by Thrusfield (1995), considering the following epidemiological data: expected prevalence of 16% based on the results of a similar study in no specific dog population from Campania region (southern Italy) (Cringoli et al. 2001); confidence interval (99%) and desired absolute precision (2%), based on the number of hunters in Campania region (n°38,611 hunters in the season 2014-2015 and assuming one dog for each hunter) (BURC 2019).

Sample collection
For each dog, blood samples were obtained through percutaneous venipuncture of cephalic or jugular veins, after 12h of fasting. The total amount of blood was immediately divided into three aliquots. The first two aliquots were placed in tubes containing potassium ethylene diamine tetra-acetic acid (EDTA) for complete blood count (CBC) and Knott test, which were carried out within 30 min of blood collection. The third aliquot was put in tubes without anticoagulant, allowed to clot and centrifuged at 908g for 15min at 4°C, in order to obtain blood serum samples that were stored at −80°C and defrosted immediately before batch analyses of haematochemical parameters.

Parasitological analysis
For each dog involved in the study, the Knott's test technique (Knott, 1939) was carried out at the parasitology laboratories of the Department of Veterinary Medicine and Animal Productions, University Federico II, Naples, to find microfilariae in blood as follows: 1 ml of whole blood was diluted with 9 ml of 2% formalin in a centrifuge tube and after centrifugation for 10 min at 1500 rpm the supernatant was discarded. Sediment sample (20 μl) was transferred to a glass slide up to complete 1 ml and examined using optical microscope at 10, 20, 40, and ×100 magnifications (Genchi et al. 2010).
In order to differentiate A. reconditum from other filarial species, blood microfilariae (D. repens, D. immitis, A. dracunculoides) were identified by morphological and morphometrical examinations. The diagnostic keys used to differentiate microfilariae species were morphology, shape of larval head and tail, and size measurements of length and width according to McCall et al. (2008). In addition, microfilariae were counted, and numbers were expressed as microfilariae/ml of blood (mf/ml blood). All the diagnostic procedures were performed with a LEICA DM 750 microscope (Germany) with digital camera; the image analysis system Leica Application Suite V 4.9 (LAS -®Leica Microsystem) was used to get microfilariae measurements and take pictures.

Clinical examination, complete blood count, and serum biochemistry
A clinical examination was performed on each A. reconditum microfilaremic dog to evaluate the presence of systemic clinical signs such as fever, lymphadenomegaly, splenomegaly, changes in mucous membrane colour, cardiorespiratory abnormalities, skin lesions, and dehydration. The nutritional status was assessed using a nine-point body condition score (BCS) system (Laflamme 1997). A complete blood count (CBC) and a basic biochemical panel were obtained on each positive animal at the clinical pathology laboratories of the Department of Veterinary Medicine and Animal Productions, University Federico II, Naples.

Statistical analysis
Factors influencing the probability for dogs of being infected by A. reconditum were explored through a mixed effects logistic regression model with binary outcome. Ninety subjects were removed from the analysis due to missing information Fig. 1 Pooled prevalence (%) of Acanthocheilonema reconditum in dogs within the European Union and adjacent countries at national level about one or more of the considered explanatory variables, for a final sample size of 2930 dogs. The effect on A. reconditum infection status (infected or uninfected) of the following variables extracted from the owners' questionnaire was examined: dog's age, gender, type of coat (short, medium, or long hair), living area (province) and environment (rural or urban), night shelter availability (yes or no), pack size, number of hunting months, type of hunted species (birds or wild mammals), travel abroad (yes or no), reported ectoparasite infestation history (yes or no), and ectoparasite treatment (yes or no). Owners' IDs were included in the model as a random factor, to account for potential covariance within packs. We first fitted a full, saturated model including all factors and obtained a minimal model through backward elimination of non-significant variables. Odds ratio (OR) estimates of the significant factors retained in the minimal model and their 95% confidence intervals (CIs) are presented.
For dogs with only A. reconditum infection, a Pearson's correlations was used to examine the association of the measured microfilarial load with the following haematological and biochemical variables: red blood cell count (RBC), haemoglobin concentration (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC) and percentage, absolute value of lymphocytes, neutrophils, eosinophils, and monocytes, and serum albumin level. All the analyses were carried out using SAS/STAT 9.4 software (Copyright © 2012, SAS Institute Inc., Cary, NC, USA).
Concerning the clinicopathological parameters, there was a significant negative correlation between microfilaremic load and the serum albumin levels (Pearson's correlation coefficient: −0.37; p=0.021) (Fig. 7). As the microfilariae number increased, the albumin level decreased linearly. For example, a dog with 100 microfilariae will have significantly lower albumin level than a dog with only microfilariae counts of two, despite the albumin value being within the laboratory reference range. There was no correlation with any other haematological or biochemical variable.

Discussion
Our study confirmed the presence of A. reconditum in hunting dogs living in Campania region of southern Italy. Of the 3020 hunting dogs tested for A. reconditum, 84 were microfilaremic with an overall prevalence of 2.8%. In a previous survey, Cringoli et al. (2001) reported a higher prevalence (16.5%; 58/351) in a no specific dog population of the same region. This discrepancy could be explained by the smaller size of the analyzed sample, the concentration of the enrolled dogs in a limited area of the Campania region (Napoli province), and the greater knowledge of the owners about the prophylaxis against ectoparasites developed in the last 20 years. Similar to our results, in a more recent survey in Molise, a small     (Lindemann and McCall 1983), it is possible that this analyzed population contained Knott's test undiagnosed cases. It is also important to underline that to date, the distribution of A. reconditum could be underestimated because it is generally identified during the search for other filarial species. Moreover, the routine use of in-clinic rapid tests for the detection of D. immitis antigens in blood samples has facilitated the diagnosis of this major filarial worm in dogs, but has likely contributed to a reduction in the opportunity to diagnose A. reconditum infections through the Knott's test that is a time-consuming procedure (Magnis et al. 2013;Evans et al. 2019). Recently, the molecular methods that allow to achieve a diagnosis at species level for A. reconditum have increased compared to testing modalities used in the past (Laidoudi et al. 2020a(Laidoudi et al. , 2020b, but as they are cannot yet be considered a routine diagnostic resource. The history of ectoparasite infestation and hunting of wild mammals are risk factors for A. reconditum in hunting dogs. These findings could be explained by the close contact that occurs between wild mammals and dogs during hunting activities, which exposes the dog to the ectoparasites infesting the prey. A recent study described C. felis and C. canis as most common infestations of wildlife, highlighting that sharing habitats between mammals of different species facilitates the ectoparasite spillover (Clark et al. 2018). The spread of filariasis within wild canid populations, such as foxes, has been well documented in Europe (Hodžić et al. 2015;Ionică et al. 2017). In central Italy, the presence of A. reconditum in fox populations reported a prevalence of 10.9% (Marconcini et al. 1996) and 9.1% (Magi et al. 2008), resulting the most common filariasis identified among investigated foxes. Furthermore, the spread of A. reconditum is related to its presence in confined populations, thus probably indicating that the infected animals may play a key role as reservoir . In fact, the latter authors during a 2-year study reported an A. reconditum prevalence of 11.2% in a confined shelter dog population, with an annual incidence of 5.9%. This illustrates how close contact with A. reconditum-infected hosts is crucial for the development of new cases, because only the adult flea stage is a competent vector for transmission. Therefore, the proximity, during hunting activities of dogs with wild animals, which are potential reservoirs of A. reconditum, could play an important epidemiological role in transmission (Diakou et al. 2016). It should also be added that dogs used for hunting mammals (mainly wild boars) are maintained in packs of at least 4-5 animals (Sgroi et al. 2020), a factor that can favour the spread of the ectoparasites among dogs reared together and thus could facilitate the transmission of A. reconditum.
There were significant differences between geographical areas, with higher prevalence in dogs from Caserta and Napoli provinces of Campania region. These results may be related to different dog management practices and to environmental factors as well. For instance, in Caserta province more than one microfilaremic dog belonged to the same owner and most microfilaremic dogs did not receive adequate ectoparasiticide treatment program during the year. In fact, the owners limited the administration of generic spot-on solutions only during the warmer period, leaving dogs exposed to ectoparasites especially during the hunting season. In Napoli province, the high presence of A. reconditum had been previously reported, suggesting a localized hotspot (Cringoli et al. 2001). Napoli province was indeed the highest risk, since A. reconditum prevalence was relatively high despite only a few dogs having an ectoparasite history, none of which hunted mammals, contrary to Caserta where these risk factors were well represented. It is important to underline that in Campania region the presence of suitable vectors is well documented: Rinaldi et al. (2007) described C. felis as the most common flea species infesting the dog population, even in urban areas, reporting that the climatic conditions would easily support the presence of fleas all year long.
Regarding the limited clinical, haematological, and serum biochemical findings generated for the 71 A. reconditum-infected dogs, this parasite appears to be minimally pathogenic. Specifically, during the clinical examination, we did not observe any skin lesions (e.g. dermatitis, alopecia and nodules), as previously reported by Brianti et al. (2012). Two A. reconditum microfilaremic dogs were reported by owners to have poor performance during hunting activity. Exercise intolerance is a clinical phenomenon that is often observed in dogs during the hunting season (Hunt et al. 2018). It can be difficult to establish the cause of episodic weakness, as it can be related to several pathological conditions, including heartworm disease, which is always considered in the differential diagnosis by clinicians. Papazahariadou et al. (1994) reported a significant correlation between episodic weakness and D. immitis microfilaraemia in hunting dogs living in Greece, but this association was not confirmed by authors for A. reconditum microfilaremic dogs.
Concerning the limited haematological variables among infected dogs, our results identified leukocytosis with eosinophilia and monocytosis. These findings are consistent with data reported during the acute and chronic phase of an experimentally induced infection . Similarly, Hashem and Badawy (2007) reported leukocytosis, with neutrophilia, eosinophilia, lymphocytosis, and monocytosis in dogs naturally infected with A. reconditum. Leukocytosis, primarily involving eosinophils, monocytes, and neutrophils, can be related to the dog's immune response to cuticular microfilarial antigens (Simon and Genchi 2001).
A finding of increased serum total proteins and total globulin levels in our study population is consistent with findings reported by Hashem and Badawy (2007), and can be explained by a chronic immune response to the parasite or perhaps to other undiagnosed infections. Acanthocheilonema reconditum microfilaremic dogs in our study had no consistent indications of liver or kidney injury, as instead previously reported by Hashem and Badawy (2007).
Hypoalbuminemia has been reported in D. immitis and D. repens infections because of liver and kidney damage due to immune-pathological and mechanical activity of microfilariae (Mircean et al. 2017). Liver injury was a speculated cause for hypoalbuminemia (Hashem and Badawy 2007); however, when hypoalbuminemia occurs in natural A. reconditum infections, it is likely due to another cause. Hypoalbuminemia was not detected by  in experimental A. reconditum study, despite documented proteinuria during the chronic infection phase. In the current study, there was a significant negative correlation between microfilaremic load and serum albumin level. This finding could be explained by the nutritional utilization by the microfilariae of amino acids from the bloodstream (Simon and Genchi 2001;Simón et al. 2012) or alternatively other mechanisms. Among microfilaremic dogs in this study, only a single animal was hypoalbuminemic, whereas the albumin concentrations of the other dogs, although negatively related to the parasite load, remained in the reference range, suggesting a body response to the presence of microfilariae but not a real pathological effect.

Conclusions
In conclusion, the present study further confirms the circulation of A. reconditum infection in southern Italy and highlights how hunting dogs represent a risk population due to their outdoor lifestyle and their close contact with wildlife. Moreover, regarding this filarial infection, further studies should be performed to better investigate epidemiological relationships between hunting dogs, wild animal populations, and the biological role of the different competent vectors sharing the same area. Finally, our data highlight that A. reconditum can be considered a minimally or nonpathogenic filarial infection in dogs. However, as some dogs may have a high microfilarial load, it is important to adhere to preventive measures against arthropod vectors to limit the infection with this filarial worm. Stanislao Ricciardi, Antonio Ricco, Giovanni Russo (ASL Salerno), Pasquale Russo, Edoardo Sanfelice di Monteforte, Giampiero Sepe, Giuseppe Sorrentino, Mario Sorrentino, Luigi Scarpato, Valerio Toscano, Alessio Vitale, Pierluigi Vitale, Antonio Zotti, and Alessandro Zullo.
Author contribution Experimental conception and design: Diego Piantedosi and Vincenzo Veneziano. Collection and analysis of samples: Laura Pacifico, Giovanni Sgroi, Benedetto Neola, and Francesco Buono. Statistical analysis: Nicola Ferrari and Claudia Romeo. Data processing and maps creation: Paolo Varuzza. Analysis and interpretation: Vincenzo Veneziano, Diego Piantedosi, Ramaswamy Chandrashekar, Melissa Beall, Laura Pacifico, Jesse Buch, and Edward B. Breitschwerdt. Paper writing: Laura Pacifico, Diego Piantedosi, Claudia Romeo, and Vincenzo Veneziano. All authors read and approved the final manuscript.
Funding Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement. The study was supported by the management committees of the hunting districts of Napoli (ATC NA), Avellino (ATC AV), Salerno (ATC SA 1), Benevento (ATC BN) and Caserta (ATC CE).
Availability of data and materials The data supporting the conclusions of this article are included within the article.

Declarations
Ethics approval and consent to participate All procedures were performed for diagnostic purposes during a routine health check, and for this reason, the study did not require an ethical approval according to European Directive 2010/63/EU; a written informed consent was obtained from the owners of the dogs included in the study.

Conflict of interest The authors declare no competing interests.
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