# Dirofilaria spp. infection in cats from the Mediterranean basin: diagnosis and epidemiology

Mariaelisa Carbonara, Livia Perles, Luigi Venco, Simona Gabrielli, Vanessa R Barrs, Guadalupe Miró, Elias Papadopoulos, Clara Lima, Emilie Bouhsira, Gad Baneth, et al.

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Mariaelisa Carbonara, Livia Perles, Luigi Venco, Simona Gabrielli, Vanessa R Barrs, et al. Dirofilaria spp. infection in cats from the Mediterranean basin: diagnosis and epidemiology. International Journal for Parasitology, 2025, 55, pp.317 - 325. 10.1016/j.ijpara.2025.01.011. hal-05200456

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# *Dirofilaria* spp. infection in cats from the Mediterranean basin: diagnosis and epidemiology ☆

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Mariaelisa Carbonara<sup>a</sup>, Livia Perles<sup>a</sup>, Luigi Venco<sup>b</sup>, Simona Gabrielli<sup>c</sup>, Vanessa R. Barrs<sup>d</sup>, Guadalupe Miró<sup>e</sup>, Elias Papadopoulos<sup>f</sup>, Clara Lima<sup>g</sup>, Emilie Bouhsira<sup>h</sup>, Gad Baneth<sup>i</sup>, Nikola Pantchev<sup>j</sup>, Roberta Iatta<sup>k</sup>, Jairo Alfonso Mendoza-Roldan<sup>a</sup>, Nicola Decaro<sup>a</sup>, Bettina Schunack<sup>l</sup>, Giovanni Benelli<sup>m</sup>, Domenico Otranto<sup>a,n,*</sup>

## ARTICLE INFO

**Article history:**

Received 17 December 2024

Received in revised form 13 January 2025

Accepted 31 January 2025

Available online 3 February 2025

**Keywords:**

Cats

Clinical signs

*Dirofilaria immitis*

*Dirofilaria repens*

Clinicopathological abnormalities

Risk factors

## ABSTRACT

*Dirofilaria immitis* and *Dirofilaria repens*, causing heartworm disease and subcutaneous dirofilariosis, respectively, are zoonotic mosquito-borne filarioids infecting a plethora of hosts including cats. Only fragmented data are available on the diagnosis and epidemiology of feline dirofilariosis. We assessed the occurrence of both nematode infections, their risk factors and clinicopathological abnormalities in cats, from six countries of the Mediterranean Basin. In addition, *Wolbachia* spp. endosymbionts were assessed in *Dirofilaria* spp.-positive animals. Blood and sera samples were obtained from cats with outdoor access from Spain (n = 354), Portugal (n = 287), Italy (n = 125), Greece (n = 116), Israel (n = 101) and France (n = 100). Cat sera were tested by both direct antigenic (SNAP test, commercial ELISA kit) and indirect antibodies (in-house ELISA) serological tools, and blood samples by real time and conventional PCR targeting *Dirofilaria* spp. DNA, followed by sequencing. A statistical analysis was run to assess the link between *Dirofilaria* spp. infection and independent variables, as well as among feline immunodeficiency virus (FIV) and/or feline leukaemia virus (FeLV) co-infections, and clinicopathological abnormalities. Overall, 3.8% (i.e., 41/1,083) cats scored positive for *Dirofilaria* spp. infection with prevalences ranging from 2% in Israel to 7.8% in Greece. Of the 41 positive cats, 16 were infected by *D. immitis* (by SNAP test and/or PCR) and two by *D. repens* (by PCR); the remaining animals were antibody-positive for *Dirofilaria* spp. using the in-house ELISA. *Wolbachia* DNA was detected in one *D. immitis*-infected cat. Nematode positivity was significantly associated with age, breed, hyporexia, dandruff, and dyspnoea. This study provides data on the prevalence of *Dirofilaria* spp. infection in cats from the Mediterranean Basin, as well as new insights on its diagnosis, revealing the importance of performing strategic chemoprophylactic treatments for cats living in areas where the infection is endemic in dogs.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)).

<footnote><sup>a</sup>Department of Veterinary Medicine, University of Bari, 70010, Bari, Italy</footnote>

<footnote><sup>b</sup>Ospedale Veterinario Città di Pavia, 27100, Pavia, Italy</footnote>

<footnote><sup>c</sup>Department of Public Health and Infectious Diseases “Sapienza”, University of Rome, 00185, Rome, Italy</footnote>

<footnote><sup>d</sup>Department of Veterinary Clinical Sciences, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon Tong 999077, Hong Kong, China</footnote>

<footnote><sup>e</sup>Animal Health Department, Veterinary Faculty, Universidad Complutense de Madrid, 28040, Spain</footnote>

<footnote><sup>f</sup>Department of Infectious and Parasitic Diseases and Pathology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124, Greece</footnote>

<footnote><sup>g</sup>Department of Biological Sciences, Microbiology Laboratory, Faculty of Pharmacy, University of Porto, 4169-007, Portugal</footnote>

<footnote><sup>h</sup>InTheres, Université de Toulouse, INRAE, ENVT, Toulouse 31076, France</footnote>

<footnote><sup>i</sup>School of Veterinary Medicine, Hebrew University, Rehovot, 76100, Israel</footnote>

<footnote><sup>j</sup>IDEXX Laboratories, Kornwestheim 70806, Germany</footnote>

<footnote><sup>k</sup>Interdisciplinary Department of Medicine, University of Bari, 70124, Bari, Italy</footnote>

<footnote><sup>l</sup>Elanco Animal Health, Monheim 40789, Germany</footnote>

<footnote><sup>m</sup>Department of Agriculture, Food and Environment, University of Pisa, 56124, Pisa, Italy</footnote>

<footnote><sup>n</sup>Department of Veterinary Clinical Sciences, City University of Hong Kong, 999077, China</footnote>

<footnote>* Nucleotide sequence data reported in this paper are available in GenBank under accession numbers PQ560497-99, PQ576422-4, PQ560500-1 and PQ576425-6.</footnote>

<footnote>* Corresponding author at: Department of Veterinary Medicine University of Bari, Str. prov. per Casamassima km 3, 70010 Valenzano, Bari, Italy.</footnote>

<footnote>E-mail addresses: mariaelisa.carbonara@uniba.it (M. Carbonara), livia.perles@uniba.it (L. Perles), luigivenco@libero.it (L. Venco), simona.gabrielli@uniroma1.it (S. Gabrielli), vanessa.barrs@cityu.edu.hk (V.R. Barrs), gmiro@ucm.es (G. Miró), eliaspap@vet.auth.gr (E. Papadopoulos), emilie.bouhsira@envt.fr (E. Bouhsira), gad.baneth@mail.huji.ac.il (G. Baneth), nikola-pantchev@idexx.com (N. Pantchev), roberta.iatta@uniba.it (R. Iatta), jairo.mendozaroldan@uniba.it (J.A. Mendoza-Roldan), nicola.decaro@uniba.it (N. Decaro), bettina.schunack@elancoah.com (B. Schunack), giovanni.benelli@unipi.it (G. Benelli), domenico.otranto@uniba.it (D. Otranto).</footnote>
# 1. Introduction

Within the family Onchocercidae, *Dirofilaria immitis* and *Dirofilaria repens* are widespread mosquito-borne filarioids primarily infecting dogs and occasionally humans, as well as other wild and domestic carnivores (Simón et al., 2022; Perles et al., 2024). Cats play a limited role in parasite circulation compared with dogs, being less commonly infected and presenting lower parasite burdens (Venco et al., 2011). Specifically, feline heartworm infection by *D. immitis* is characterized by transient microfilaremia or an absence of microfilaremia (i.e., occult infection) and aberrant parasite location (McCall et al., 1992). However, in endemic geographical areas where chemoprophylaxis against dirofilariosis is systematically conducted in dogs, alternative mammals such as foxes or, to a lesser extent cats, may contribute to maintaining the endemicity of the infection (Ciuca et al., 2020; Panarese et al., 2021; Gomes-de-Sá et al., 2022; Villanueva-Saz et al., 2024). Overall, the prevalence of *D. immitis* infection in cats ranges from 9% to 18% of that observed in dogs from the same areas (Venco et al., 2011). However, cases are probably underestimated as infected cats often have subclinical infections or develop chronic/non-specific signs (e.g., mild to moderate respiratory disorders, anorexia, lethargy, weight loss, vomiting) and laboratory alterations (e.g., eosinophilia), even several months after infection (Pennisi et al., 2020). In addition, when nematodes die in the pulmonary arteries, they cause severe inflammation and thromboembolism, resulting in an acute heartworm-associated respiratory disease (HARD), which is often fatal (Venco et al., 2015; Garrity et al., 2019; Pennisi et al., 2020; Nelson and Johnson, 2024).

Due to its zoonotic potential in the Old World, there is an increasing interest in feline subcutaneous dirofilariosis caused by *D. repens* (Otranto et al., 2011; Simón et al., 2022; Perles et al., 2024), although there have been a few studies conducted in Europe, which recorded a prevalence ranging from 0.7% in Poland to 1.6% in Italy ESDA, 2021). From a clinical perspective, feline subcutaneous dirofilariosis has limited relevance, usually being subclinical or characterized by mild to moderate cutaneous lesions (e.g., nodules, pruritus, erythema) (Manzocchi et al., 2017). As a result, *D. repens* worms are only occasionally retrieved from cats, mainly following cytology of skin lesions (Ciuca et al., 2020; Panarese et al., 2021). Considering the above, the detection of *D. immitis* and *D. repens* infections in cats represents a challenging task and may be hampered by false negative results, highlighting the importance of employing both direct and indirect tests targeting, respectively, *Dirofilaria* spp. antigens (e.g., ELISA tests towards *D. immitis* female antigens, or PCR targeting blood-circulating microfilariae of *Dirofilaria* spp.) and antibodies (e.g., indirect ELISA). Specifically, in the absence of circulating female adult antigens and/or microfilariae, antibody detection indicates a cat's exposure, which can represent a good approach for *Dirofilaria* spp. infection diagnosis in animals living in or travelling to endemic areas (Venco et al., 2015; ESDA, 2021). However, for clinical diagnosis of feline dirofilariosis, thoracic radiography and echocardiography are crucial (Litster and Atwell, 2008). In addition, the detection of a *Wolbachia* endosymbiont has been used for its positive predictive value in diagnoses of dirofilarioses in both humans (Manoj et al., 2021; Mendoza-Roldan et al., 2021) and animal hosts such as cats (Turba et al., 2012). Species of the genus *Wolbachia* include Gram-negative bacteria, essential for the survival of *Dirofilaria* spp., located in the female reproductive tract, as well as within lateral chords of both male and female nematodes, in all developmental stages, which makes these bacteria detectable even during occult infections (Kramer et al., 2003; Kozek, 2005; Laidoudi et al., 2020). Accordingly, *Wolbachia* spp. were detected by conventional PCR (cPCR) targeting the 16S rRNA gene, in an asymptomatic human patient infected by *D. repens* (Mendoza-Roldan et al., 2021).

Overall, fragmented data are available on the geographical distribution, epidemiology and risk factors of feline dirofilariosis in the Old World. Furthermore, diagnostic data are scarce due to the absence of systematic epidemiological surveys employing the same diagnostic procedures. To fill this gap, we assessed the occurrence of *D. immitis* and *D. repens* infections, by means of all laboratory diagnostic techniques available (i.e., SNAP test for antigen detection, indirect ELISA for antibodies and real time PCR, quantitative PCR (qPCR) for DNA), their associated risk factors and clini-copathological abnormalities in domestic feline populations from six countries of the Mediterranean Basin, which were enrolled in a previous multi-center study on feline leishmaniosis (Carbonara et al., 2024). In addition, we assessed the presence of *Wolbachia* spp. endosymbionts in animals positive for *Dirofilaria* spp. infection.

# 2. Materials and methods

## 2.1. Ethics statement

Written consent for patient enrolment was obtained from owners or people in charge of animal protection associations. Animals were handled with regard for their well-being and the protocol of this study was approved by the ethical committees of the Complutense University of Madrid, Spain (Prot. 60-29102019); and the Organism Responsible for Animal Welfare (ORBEA) from the Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Portugal; the Department of Veterinary Medicine, University of Bari, Italy (Prot. Uniba 7/17); the Aristotle University of Thessaloniki, Greece (Prot. 192746/2022) and the Koret School of Veterinary Medicine Teaching Hospital, Hebrew University of Jerusalem, Israel (Prot. KSVMVTH/7_2018).

## 2.2. Study locations and cat sampling

Domestic cats (n = 1,083) were sampled as part of a previous multicenter study on feline leishmaniosis (Carbonara et al., 2024). Briefly, blood and sera samples were obtained from predominantly free roaming or shelter-housed cats living in southern Mediterranean countries including Spain (n = 354), Portugal (n = 287), Italy (n = 125), Greece (n = 116), Israel (n = 101) and France (n = 100). Inclusion criteria were a history of outdoor access and no previous treatment with ectoparasiticides or repellents for at least 6 months prior to sampling. At enrolment, animal data (i.e., age, breed, sex), together with housing conditions and the geographical origin (city/town, zip code) were recorded in individual files. Clinical data, available for n = 882 cats, were categorized as follows: systemic clinical signs (i.e., asthenia, fever, hepatomegaly, hyporexia, jaundice, lymphadenomegaly, pale mucous membranes, weight loss); cutaneous lesions (i.e., alopecia, dandruff, nodules/haemorrhagic cysts, squamous cell carcinoma, ulcers/ crusts/scales), ocular signs (i.e., blepharitis, keratitis, conjunctivitis, uveitis), oral signs (i.e., gingivostomatitis, mouth ulcers), gastrointestinal signs (i.e., diarrhoea, vomiting), respiratory signs (i.e., coughing, dyspnoea, nasal discharge, ocular discharge), and clinical signs caused by chronic kidney disease (i.e., cachexia, hyporexia, polyuria and polydipsia, weight loss). In addition, for 419 cats, health status and laboratory parameters including complete blood cell count (CBC) and biochemical profile (i.e., creatinine, urea, ala-nine aminotransferase (ALT)), including serum protein electrophoresis (i.e., albumin, total proteins, total globulins, and albumin/globulins ratio (A/G)) were recorded in individual patient records. Cats enrolled were grouped according to their age as: young (from 7 months to 6 years), mature (from 6 years and 1 month old to 10 years) and senior (>10 years).
## 2.3. Antigenic and serological testing

Serum samples (n = 1,083) were tested for the detection of *D. immitis* female antigens using the commercial ELISA kit (SNAP® Heartworm RT Test, IDEXX, Westbrook, ME 04092 USA), following the manufacturer's instructions. The SNAP test used had 98.9% (95% Confidence Interval (CI): 93.8–99.9) sensitivity (Se) and 100% (95% CI: 78–100) specificity (Sp) (Genchi et al., 2018). Sera were not preheated to exclude immune complex formation (Beall et al., 2017) due to the low amount of volume. Additionally, the same sera were further tested to assess exposure to *Dirofilaria* spp. using an in-house ELISA based on somatic antigens of adult *D. immitis*, with a final concentration of 0.4 µg/µl (Perles et al., 2025). Sera were tested in solid-phase ELISA at a dilution 1:200 and goat anti-cat-IgG conjugated to horseradish peroxidase (Sigma-Aldrich, St. Louis, MO 63118, USA) was used as a secondary antibody at 1:20,000 dilution. The optical density (O.D.) was measured at 450 nm (Easy-Reader, Bio-Rad, Hercules, CA, USA). The cut-off point (O.D. = 1.15) was established by calculating the mean value + 3 S.D. of 100 control serum samples from seronegative and PCR-negative cats living in a non-endemic region. The in-house ELISA employed had 89% Se and 98% Sp (Perles et al., 2025).

## 2.4. Molecular analysis

DNA was extracted from 200 µl of whole blood using a commercial kit (QIAamp DNA Blood Tissue, Qiagen, Hilden, Germany) according to the manufacturer's instructions and was analysed for the detection of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) proviral DNA by cPCR (Endo et al., 1997; Stiles et al., 1999). Whole blood DNA samples were tested in the current study by a duplex quantitative real-time PCR (dqPCR) assay for the detection of *D. immitis* (cytochrome c oxidase subunit 1 gene, *cox1* gene) and *D. repens* (second internal transcribed spacer) DNA (both microfilariae and adults' circulating genomic DNA) using primers, probes, and protocols described elsewhere (Latrofa et al., 2012). The above dqPCR had high Se, being capable of detecting small amounts of genomic DNA (2.5 pg/µl and $3 \times 10^{-1}$ pg/µl for *D. immitis* and *D. repens*, respectively). The Sp was established by melting-curve analyses as follow: melting curve temperature = 75.7 ± 0.3 °C for *D. immitis* DNA, melting curve temperature = 70 ± 0.7 °C for *D. repens* (Latrofa et al., 2012). In addition, positive samples on dqPCR were tested by two cPCRs targeting *cox1* (Pradeep et al., 2019) and 12S rDNA genes (Otranto et al., 2011), respectively, to obtain reference sequences. The same positive samples were tested by qPCR targeting theftsZ gene of *Wolbachia* associated with *D. immitis*, as previously described (Louzada-Flores et al., 2022). cPCR products were visualized using a UV transilluminator after electrophoresis on a 2% agarose gel stained with red dye. Amplicons of the expected size were purified and sequenced in both directions using the Big Dye Terminator v3.1 chemistry in a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in an automated sequencer (ABI-PRISM 377). For all PCR runs, positive (i.e., DNA extracted from a blood sample of a dog molecularly positive for *D. immitis* and from another dog positive for *D. repens*) and negative controls (DNA extracted from blood samples of negative healthy cats) were included.

Sequences were edited using BioEdit v7.2.5 and compared with other *Dirofilaria* spp. sequences available on GenBank. For phylogenetic inference, sequences from the present study were aligned with those retrieved from GenBank using the MAFFT software version 7 (Katoh et al., 2018). The best evolutionary model was chosen under the Akaike Information Criterion (AIC) using the CIPRES gateway. Maximum likelihood phylogenetic analyses with 8000

bootstraps were performed using the iqTree gateway (Trifinopoulos et al., 2016). The phylogenetic tree edition and rooting (outgroup) were performed using TreeGraph 2.0 beta software (Stöver and Müller, 2010).

## 2.5. Statistical analysis

The cumulative prevalence of *Dirofilaria* spp. infection was calculated by summing the positivity to at least one test employed. Apparent prevalences were recorded, considering that cats were scored as *D. immitis*-positive when positive to a SNAP test and/or by dpPCR (i.e., melting curve temperature = 75.7 ± 0.3 °C); similarly, they were considered *D. repens*-positive when positive by dpPCR (i.e., with melting curve temperature = 70 ± 0.7 °C) (Latrofa et al., 2012). Given the cross-reaction between *D. immitis* and *D. repens* on indirect ELISA testing (Mendoza-Roldan et al., 2021; Pěkacz et al., 2024; Perles et al., 2025), cats positive only to in-house ELISA were considered as *Dirofilaria* spp. seropositive. The true prevalence retrieved by in-house ELISA and a SNAP test were calculated using the formula: true Prevalence (TP) = apparent prevalence (AP) + (Sp - 1) / Se + (Sp - 1) (Dohoo et al., 2009). It was not possible to calculate the true prevalence of the dqPCR given that the PCR performances for this assay are expressed as amounts of genomic DNA detectable (i.e., 2.5 pg/µl and $3 \times 10^{-1}$ pg/µl for *D. immitis* and *D. repens*, respectively) for the Se, and melting curve temperatures for the Sp. The locations of *Dirofilaria*-positive cats were georeferenced using a geographical information system (GIS) program (QGIS software, Buenos Aires version).

Differences in cumulative *Dirofilaria* spp. positivity in cats of different sex, age, housing condition, breed, and country were analysed using a generalized linear mixed model with a binomial error structure (1 = cumulative positivity, 0 = negativity) and individual ID as a random effect. A probability level of P < 0.05 was used to assess the significance of differences among values. Contingency analyses assessing the potential relationships between *Dirofilaria* spp. positivity (yes/no), clinical signs, and laboratory alterations were conducted.

Further contingency analyses were carried out to assess the potential relationships between *Dirofilaria* spp. positivity and FIV/FeLV or *Leishmania infantum* positivity, the latter data being retrieved from the study on feline leishmaniasis by Carbonara et al. (2024). Statistical analysis was conducted using JMP Pro 17.0.0 (SAS Institute, Cary, NC, USA).

## 2.6. Data accessibility

The raw dataset supporting the results of this study is available at https://doi.org/10.17632/4rzyjrrczv.1

## 3. Results

Overall, 3.8% (i.e., 41/1,083, 95% CI: 2.8–5.1) cats scored positive for *Dirofilaria* spp. on at least one of the three tests used, with prevalences ranging from 2% in Israel (i.e., 2/100, 95% CI: 0.5–7) and up to 7.8% in Greece (i.e., 9/116; 95% CI: 4.1–14) (Table 1, Fig. 1). Out of the *Dirofilaria* spp.-positive cats (n = 41), 16 were positive for *D. immitis* and two for *D. repens*; the remaining animals (n = 23) were in-house ELISA seropositive for *Dirofilaria* spp. Out of 16 *D. immitis*-positive animals, 69% (n = 11) tested positive by SNAP test and 43.8% (n = 7) by dqPCR, with cycle threshold (CT) values ranging from 29 to 37; two cats tested *D. immitis*-positive on both SNAP and dqPCR. In addition, three animals positive by SNAP test and five by the dqPCR for *D. immitis* were in-house ELISA-negative. Both *D. repens*-positive cats tested positive on dqPCR and in-house ELISA.
<caption>**Table 1**</caption>

Comparison of serological (antibody-positive by in-house ELISA), antigenic (SNAP test) and molecular (duplex quantitative real-time PCR ((dqPCR)) prevalence (apparent prevalence (AP); true prevalence (TP)) of *Dirofilaria* spp. infections with animal data.

<table><thead><tr><th>Variables</th><th>Total cats<br>1083<br>n (%)</th><th>In-house ELISA positive<br>n (AP, TP)</th><th>SNAP test<br>positive<br>n (AP, TP)</th><th>dqPCR<br>positive<br>n (%)</th><th>D. immitis<br>positive<br>n (%)<sup>a</sup></th><th>D. repens<br>positive<br>n (%)<sup>b</sup></th><th>Total positive<br>n (%)</th></tr></thead><tbody><tr><td><strong>Age</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Youngs</td><td>961 (88.7)</td><td>28 (2.9, 1)</td><td>9 (0.9, 0.9)</td><td>8 (0.8)</td><td>14 (1.5)</td><td>2 (0.2)</td><td>36 (3.7)</td></tr><tr><td>Adults</td><td>71 (6.5)</td><td>4 (5.6, 4.1)</td><td>1 (1.4, 1.4)</td><td>0</td><td>1 (1.4)</td><td>0</td><td>4 (5.6)</td></tr><tr><td>Seniors</td><td>51 (4.7)</td><td>1 (2, 0)</td><td>1 (2, 2)</td><td>1 (2)</td><td>1 (2)</td><td>0</td><td>1 (2)</td></tr><tr><td><strong>Gender</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Female</td><td>543 (50)</td><td>13 (2.4, 0.5)</td><td>3 (0.5, 0.5)</td><td>2 (0.4)</td><td>5 (0.9)</td><td>0</td><td>17 (3.1)</td></tr><tr><td>Male</td><td>540 (50)</td><td>20 (3.7, 1.5)</td><td>8 (1.5, 1.5)</td><td>7 (1.3)</td><td>11 (2)</td><td>2 (0.4)</td><td>24 (4.4)</td></tr><tr><td><strong>Housing condition</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sheltered/free roaming</td><td>862 (79.6)</td><td>25 (2.9, 1)</td><td>11 (1.3, 1.3)</td><td>9 (1)</td><td>16 (1.9)</td><td>2 (0.2)</td><td>33 (3.8)</td></tr><tr><td>Owned</td><td>221 (20.4)</td><td>8 (3.6, 1.8)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>8 (3.6)</td></tr><tr><td><strong>Breed</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>European</td><td>1053 (97.1)</td><td>33 (3.1, 1.3)</td><td>11 (1, 1)</td><td>9 (0.8)</td><td>16 (1.5)</td><td>2 (0.1)</td><td>41 (3.9)</td></tr><tr><td>Non-European</td><td>30 (2.9)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td><strong>Country</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Spain</td><td>354 (32.7)</td><td>11 (3.1, 1.3)</td><td>1 (0.3, 0.3)</td><td>5 (1.4)</td><td>5 (1.4)</td><td>1 (0.3)</td><td>15 (4.2)</td></tr><tr><td>Italy</td><td>125 (11.5)</td><td>4 (3.2, 1.4)</td><td>4 (3.2, 3.2)</td><td>3 (2.4)</td><td>5 (4)</td><td>0</td><td>6 (4.8)</td></tr><tr><td>Greece</td><td>116 (10.7)</td><td>8 (6.9, 5.6)</td><td>3 (2.6, 2.6)</td><td>0</td><td>3 (2.6)</td><td>0</td><td>9 (7.8)</td></tr><tr><td>Israel</td><td>101 (9.3)</td><td>2 (2, 0)</td><td>0</td><td>0</td><td>0</td><td>0</td><td>2 (2)</td></tr><tr><td>Portugal</td><td>287 (26.5)</td><td>5 (1.7, -0.3)</td><td>3 (1, 3)</td><td>0</td><td>3 (1)</td><td>0</td><td>6 (2)</td></tr><tr><td>France</td><td>100 (9.2)</td><td>3 (3, 1.1)</td><td>0</td><td>1</td><td>0</td><td>1 (1)</td><td>3 (3)</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Map of study areas (i.e., Portugal, Spain, France, Italy, Greece, Israel) with sampled provinces (dark-colored), showing the number of samples according to their *Dirofilaria* spp. positivity. Stars indicate areas discussed in the text.</figcaption></figure>

<footnote>a *Dirofilaria immitis* positivity: SNAP test-positive and/or dqPCR positive with melting curve temperature = 75.7 ± 0.3 °C.</footnote>

<footnote>b *Dirofilaria repens* positivity: dqPCR-positive with melting curve temperature = 70 ± 0.7 °C.</footnote>
The apparent prevalences retrieved by in-house ELISA and SNAP test were 3% (i.e., 33/1,083) and 1% (i.e., 11/1,083), respectively; the true prevalences were 1.1% and 1%, respectively.

The distribution of *Dirofilaria* spp.-positive cats by geographical area is depicted in Fig. 1.

The phylogenetic relationships of *cox1* (Fig. 2A) and 12S rDNA genes (Fig. 2B) showed similar topologies, with the three sequences of *D. immitis* from cats in Italy (submitted to GenBank under accession numbers **PQ560497-99; PQ576422-4**), clustering into a separate *D. immitis* clade, together with those from canine isolates, from France and Italy. As for *D. repens*, the two sequences obtained from Spain (submitted to GenBank under accession numbers **PQ560500, PQ576425**) and France (submitted to GenBank under accession numbers **PQ560501, PQ576426**) formed a separate clade with those from humans and dogs living in Italy (**OP024281**), France (**MT230098**) and Russia (**KM205377**). The two *cox1* *D. repens* sequences herein obtained diverged by two nucleotides, whereas all three *D. immitis* sequences were identical. *Wolbachia* DNA was detected in only one *D. immitis*-infected animal (i.e., 1/16), which scored positive by in-house ELISA, SNAP test and dqPCR (CT: 29, the lowest herein detected).

The prevalences of *Dirofilaria* spp. infection in relation to the diagnostic test employed, animal data (i.e., age, gender, housing condition, breed, and geographical area) and clinicopathological information are reported in Tables 1 and 2.

The risk of *Dirofilaria* spp. infection in cats was significantly associated with age (i.e., *F* = 5.430, degrees of freedom (*df*) = 2, *P* = 0.005) and breed (i.e., common European breed, *F* = 7.290, *df* = 1, *P* = 0.007), while housing condition (*F* = 0.483, *df* = 1, *P* = 0.487), gender (*F* = 0.621, *df* = 1, *P* = 0.430), and country (*F* = 2.100, *df* = 5, *P* = 0.063) were not significant. No significant association between *Dirofilaria* positivity and FIV (*χ*² = 0.016, *df* = 1, *P* = 0.900), FeLV (*χ*² = 1.131, *df* = 1, *P* = 0.288) or *Leishmania* positivity (*χ*² = 0.013, *df* = 1, *P* = 0.910) was detected (Table 2). Out of 41 positive cats, health status data were available for 40 animals, of which 11 (27.5%) presented with at least one clinical sign, with systemic signs (i.e., 15%, 6/40) and cutaneous lesions (i.e., 15%, 6/40) being the most common. Specifically, *Dirofilaria* spp.

positivity was significantly associated with hyporexia (*χ*² = 4.122, *df* = 1, *P* = 0.042), dandruff (*χ*² = 4.269, *df* = 1, *P* = 0.039), and dyspnoea (*χ*² = 3.949, *df* = 1, *P* = 0.047) (Table 2). CBC, biochemical profile and electrophoretogram were available for 17 positive cats, of which 100% presented at least one clinicopathological alteration. Leucocytosis (i.e., 47%), hyperglobulinemia (i.e., 47%), decreased A/G ratio (i.e., 47%) and hypoalbuminemia (i.e., 41.2%) were the most frequent alterations recorded (Table 2). However, none of these were significantly associated with *Dirofilaria* spp. infection.

Overall, of the 41 *Dirofilaria* spp.-positive cats, eight cats were also positive for *L. infantum*, of which, five were *Dirofilaria* spp. seropositive and three *D. immitis*-infected (i.e., positive by SNAP test), the latter being free roaming, young aged, domestic short hair, and FIV/FeLV-negative. Specifically, CAT 1 was a female animal from Portugal that had anaemia, leucocytosis and increased ALT activity. CAT 2 and CAT 3 were both male and from Greece, with CAT 2 showing pale mucous membranes, skin nodules and respiratory signs (i.e., dyspnoea, nasal and ocular discharge) while CAT 3 had only cutaneous signs (i.e., nodules and scaling).

The *Dirofilaria* spp. sequences generated in this study were deposited in the GenBank database (i.e., *Dirofilaria immitis* from cats in Italy: accession numbers **PQ560497-99, PQ576422-4**; *D. repens* from Spain: accession numbers **PQ560500, PQ576425**; *D. repens* from France: accession numbers **PQ560501, PQ576426**).

## 4. Discussion

Data from this study demonstrate that *Dirofilaria* spp. infection circulates among cats from countries of the Mediterranean Basin, where it is endemic in dogs. However, considering that no imaging investigations (e.g., thoracic radiography and echocardiography) were performed on enrolled animals, future studies should provide more in-depth analysis on clinical aspects of feline dirofilariosis, taking imaging data into consideration.

Overall, the cumulative prevalence of *D. immitis* and *D. repens* recorded by direct and indirect diagnostic approaches (i.e., 3.8%) is similar to the average prevalence (i.e., 5.2%), according to that

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 2.** Phylogenetic trees, inferred by maximum likelihood inference, of the *Dirofilaria* spp. sequences herein obtained using the cytochrome c oxidase subunit 1, *cox1* (A) (accession numbers PQ560497-PQ560501) and 12S rDNA (B) (accession numbers PQ576422-PQ576426) genes. Sequences from the present study are highlighted in bold and the lengths of the branches are proportional to the evolutionary differences. *Ascaris lumbricoides* was used as an outgroup and numbers at nodes indicate bootstraps values.</figcaption></figure>
<table><caption>**Table 2**
Health status (A), laboratory parameters (B), retrovirus infections (feline immunodeficiency virus (FIV); feline leukaemia virus (FeLV)) (C) and *Leishmania infantum* prevalence (D) in the feline population studied.</caption><thead><tr><th></th><th>Total cats<br>n (%)</th><th>Total positive cats<br>n (%)</th></tr></thead><tbody><tr><td><strong>(A)</strong></td><td></td><td></td></tr><tr><td><strong>Health status data</strong></td><td>882</td><td>40</td></tr><tr><td>Clinical signs</td><td></td><td></td></tr><tr><td>Yes</td><td>271 (30.7)</td><td>11 (27.5)</td></tr><tr><td>No</td><td>611 (69.3)</td><td>29 (72.5)</td></tr><tr><td>Systemic signs</td><td>172 (19.5)</td><td>6 (15)</td></tr><tr><td>Skin lesions</td><td>86 (9.7)</td><td>6 (15)</td></tr><tr><td>Ocular signs</td><td>41 (4.6)</td><td>0</td></tr><tr><td>Oral signs</td><td>36 (4)</td><td>1 (2.5)</td></tr><tr><td>Gastrointestinal signs</td><td>47 (5.3)</td><td>2 (5)</td></tr><tr><td>Respiratory signs</td><td>105 (11.9)</td><td>3 (7.5)</td></tr><tr><td>Urinary signs</td><td>17 (1.9)</td><td>1 (2.5)</td></tr><tr><td><strong>(B)</strong></td><td></td><td></td></tr><tr><td><strong>Laboratory parameters data</strong></td><td>419</td><td>17</td></tr><tr><td>Clinicopathological alterations</td><td></td><td></td></tr><tr><td>Yes</td><td>410 (97.8)</td><td>17 (100)</td></tr><tr><td>No</td><td>9 (2.2)</td><td>0</td></tr><tr><td>Haematology</td><td></td><td></td></tr><tr><td>Haematocrit (28-43%)</td><td></td><td></td></tr><tr><td>High</td><td>71 (16.9)</td><td>2 (11.8)</td></tr><tr><td>Low</td><td>89 (21.2)</td><td>3 (17.6)</td></tr><tr><td>Normal</td><td>259 (61.8)</td><td>12 (70.6)</td></tr><tr><td>Leukocytes (5.5-12*1000/L)</td><td></td><td></td></tr><tr><td>High</td><td>196 (46.8)</td><td>8 (47)</td></tr><tr><td>Low</td><td>30 (7.2)</td><td>2 (11.8)</td></tr><tr><td>Normal</td><td>193 (46)</td><td>7 (41.2)</td></tr><tr><td>Platelets (130-400*100,000/ul)</td><td></td><td></td></tr><tr><td>High</td><td>64 (15.3)</td><td>1 (5.8)</td></tr><tr><td>Low</td><td>74 (17.7)</td><td>1 (5.8)</td></tr><tr><td>Normal</td><td>281 (67)</td><td>15 (88.2)</td></tr><tr><td>Biochemistry</td><td></td><td></td></tr><tr><td>Total proteins (5.8-7.7 g/dL)</td><td></td><td></td></tr><tr><td>High</td><td>137 (32.7)</td><td>5 (29.4)</td></tr><tr><td>Low</td><td>14 (3.3)</td><td>1 (5.9)</td></tr><tr><td>Normal</td><td>268 (64)</td><td>11 (64.7)</td></tr><tr><td>Albumins (2.8-3.7 g/dL)</td><td></td><td></td></tr><tr><td>High</td><td>77 (18.4)</td><td>1 (5.9)</td></tr><tr><td>Low</td><td>123 (29.3)</td><td>7 (41.2)</td></tr><tr><td>Normal</td><td>219 (52.3)</td><td>9 (52.9)</td></tr><tr><td>Total globulins (2.9-4.3 g/dL)</td><td></td><td></td></tr><tr><td>High</td><td>165 (39.4)</td><td>8 (47)</td></tr><tr><td>Low</td><td>45 (10.7)</td><td>1 (6)</td></tr><tr><td>Normal</td><td>209 (49.9)</td><td>8 (47)</td></tr><tr><td>A/G (0.6-1.3)</td><td></td><td></td></tr><tr><td>High</td><td>50 (12)</td><td>1 (6)</td></tr><tr><td>Low</td><td>113 (27)</td><td>8 (47)</td></tr><tr><td>Normal</td><td>256 (61)</td><td>8 (47)</td></tr><tr><td>Urea (29-60 mg/dL)</td><td></td><td></td></tr><tr><td>High</td><td>64 (15.4)</td><td>1 (5.9)</td></tr><tr><td>Low</td><td>120 (28.6)</td><td>3 (17.6)</td></tr><tr><td>Normal</td><td>235 (56)</td><td>13 (76.5)</td></tr><tr><td>Creatinine (0.93-1.7 mg/dL)</td><td></td><td></td></tr><tr><td>High</td><td>83 (19.8)</td><td>5 (29.4)</td></tr><tr><td>Low</td><td>113 (27)</td><td>5 (29.4)</td></tr><tr><td>Normal</td><td>223 (53.2)</td><td>7 (41.2)</td></tr><tr><td>Alanine transaminase (ALT) (33-70 UI/L)</td><td></td><td></td></tr><tr><td>High</td><td>150 (35.8)</td><td>6 (35.3)</td></tr><tr><td>Low</td><td>50 (11.9)</td><td>0</td></tr><tr><td>Normal</td><td>219 (52.3)</td><td>11 (64.7)</td></tr><tr><td><strong>(C)</strong></td><td></td><td></td></tr><tr><td><strong>Retrovirus infection data</strong></td><td>1083</td><td>35</td></tr><tr><td>FIV positivity</td><td>74 (7)</td><td>3 (8.6)</td></tr><tr><td>FeLV positivity</td><td>28 (2.7)</td><td>0</td></tr><tr><td>FIV and FeLV positivity</td><td>1 (0)</td><td>0</td></tr><tr><td><strong>(D)</strong></td><td></td><td></td></tr><tr><td><strong>L. infantum data</strong></td><td>1083</td><td>41</td></tr><tr><td>L. infantum-positive</td><td>204 (18.8)</td><td>8 (20)</td></tr><tr><td>L. infantum-negative</td><td>879 (81.2)</td><td>33 (80)</td></tr></tbody></table>

calculated from data in the literature from Greece (9.4%; Diakou et al., 2019), Italy (6.7%; Genchi et al., 2008), Portugal (4.1% mean value; Maia et al., 2015; Neves et al., 2020) and Spain (i.e., 0.5%; Montoya-Alonso et al., 2022). Studies in the literature mainly refer to canine heartworm disease with scant information available on cats from the Old World (Montoya-Alonso et al., 2024), as demonstrated by the absence of any known study in Israel or France. None of the previously reported studies on *D. immitis* infection in cats simultaneously compared serological, antigenic and molecular tests. By examining results for each country, the prevalence of *D. immitis* herein recorded in cats from Greece (2.6%, 95% CI: 0.9-7.13) is lower than that previously reported (Diakou et al., 2019). However, the prevalence in the Macedonia region, northern Greece (i.e., 3/32, 9.4%, 95% CI: 3.2-24, Diakou et al., 2019) overlaps that herein recorded in Kythnos island (i.e., 4/30, 13%, 95% CI: 5.3-29.7), as also suggested by the similar 95% CIs. Analogously, the *D. immitis* infection rate in cats from Italy (i.e., 4%, 5/125, 95% CI: 1.7-9) is lower than that formerly described from specific regions of the country (Pavia, North Italy, 6.3%, 95% CI: 5.6-7.9, Genchi et al., 2008), highly endemic for the infection in dogs (Genchi et al., 2005). Nonetheless, comparing data in the same Italian province (i.e., Lombardy), the prevalence in cats is even higher (13%, 3/23; 95% CI: 4.5-32) than the above (6.3%, 131/1962, 95% CI: 5.6-7.9, Genchi et al., 2008), demonstrating the importance of a careful examination of data at the regional level, as well as the relevance of considering the population size that may influence the prevalence comparison, as suggested by the different 95% CI ranges.

Similarly, an overall lower prevalence of cats infected by *D. immitis* was detected across Portugal (i.e., 1%, 3/287, 95% CI: 0.4-3) compared with the Algarve region (i.e., 4.8%, 13/271, 95% CI: 2.8-8, Maia et al., 2015), which is highly endemic for canine *D. immitis* infection (i.e., 8.8-17%; Cardoso et al., 2012; Esteves-Guimarães et al., 2024). The absence of cats from the Algarve region in the population of the current study represents one of its limitations. Other factors potentially influencing *Dirofilaria* infection prevalence in cats include the composition of the population enrolled. Accordingly, the 1.4% *D. immitis* rate (5/354, 95% CI: 0.6-3.3) in cats from Spain is higher than that previously recorded across the country (i.e., 0.5%, 35/6,588, 95% CI: 0.4-0.7, Montoya-Alonso et al., 2022), probably because almost all animals herein tested (i.e., 98.9%) were shelter-housed/free roaming and not owned (see Montoya-Alonso et al., 2022).

All the above data emphasize the importance that specific epidemiological contexts play in favouring parasite circulation such as in the case of islands where high *D. immitis* prevalence was detected in cats from Ibiza, Spain (i.e., 8/40, 20%, 95% CI: 10-35) and Kythnos, Greece (i.e., 4/30, 13%, 95% CI: 5.3-30) (Fig. 1). Due to their particular epidemiological features, in terms of hosts (e.g., stable pet populations) and vector circulation (e.g., suitably humid climatic conditions for mosquito development), Mediterranean islands may represent optimal spots for the development of *Dirofilaria* spp. infection. Interestingly, the two islands of Ibiza and Kythnos are included between the 35° and the 38° parallels, where Linosa Island (southern Italy) also lies and is the southernmost hyperendemic focus of *Dirofilaria* spp. infection in Europe, with prevalences of up to 59% in dogs and 17.6% in cats (Brianti et al., 2022).

Although no *Dirofilaria* spp. infection surveys have been conducted in cats from France and Israel, the antibody positivity by in-house ELISA recorded in these two areas (i.e., 3%, 95% CI: 1-8.4, in France; 2%, 95% CI: 0.5-7 in Israel) confirms *Dirofilaria* spp. presence, as indicated by previous reports in dogs (i.e., France: Tahir et al., 2017; Grosbois, 2023; Israel: Baneth et al., 2002; Mazaki-Tovi et al., 2016; Pipano, 2022).
It has been estimated that the prevalence of *D. immitis* infection in cats may range from 9% to 18% of the prevalence in dogs from the same regions (Venco et al., 2011), highlighting the importance of dogs in maintaining the infection in a given region. A similar epidemiological scenario was described for *L. infantum* infection among canine and feline populations with a prevalence in cats being about half of that in dogs (Otranto et al., 2017).

However, despite an overall reduction in canine heartworm infections recorded in the last 24 years in Italy, mostly due to canine chemoprophylaxis (Otranto et al., 2009; Mendoza-Roldan et al., 2020), cats from northern Italy remain the most frequently infected compared with other Italian provinces. In such epidemiological circumstances, where strategic treatments of dog populations are expected, *Dirofilaria* spp. infection should be monitored mainly in animals other than owned dogs, that may maintain the parasite life cycle (e.g., stray and shelter dogs, foxes and Iberian wolves, as wildlife reservoirs), usually not being treated (Gomes-de-Sá et al., 2022).

The overall exposure to *Dirofilaria* spp. infection detected by in-house ELISA accounts for approximately three times the prevalence retrieved by antigenic/molecular tests (i.e., 3% versus 0.9%). This ratio was expected and previously discussed in different epidemiological contexts of the New World (Murillo et al., 2023), supporting the notion that cats are exposed to *Dirofilaria* spp. infection but only a small proportion of them develop the disease, with the presence of at least one adult nematode that is detectable by antigen-based tests (Miller et al., 2000; Lee and Atkins, 2010). Indeed, it is estimated that only 10–20% of cats testing positive for *Dirofilaria* spp. antibodies harbour mature nematodes at the pulmonary arteries (Miller et al., 2000). This is also supported by the fact that most *Dirofilaria* spp.-positive cats scored negative for *Wolbachia* DNA, suggesting that cats may have a low parasitic burden, as also observed by the high CT values recorded by dqPCR. In addition, even though not evaluated in the present study by Knott's test, it is rare for cats to have circulating microfilariae, being the main source of *Wolbachia* spp. in blood (Louzada-Flores et al., 2022). Therefore, serological tests targeting antibodies against *Wolbachia* surface proteins (Morchón et al., 2004) might be more useful than molecular tools for early detection of the infection in patients with compatible signs. However, the seropositivity detected in cats negative by SNAP tests might be due to previous exposure, to a male-only infection or to the occurrence of immune-complexes, which made antigens undetectable (Hoch and Strickland, 2008). Conversely, the finding of seronegative cats that are SNAP or molecularly positive for *Dirofilaria* spp. can be related to at least three immunological scenarios: (i) an early infection before the development of heartworm disease, in which antibodies usually start to be detectable 3–6 months after infection; (ii) a late infection with declining antibody levels, which usually occurs approximately 1 year after infection; (iii) immunocompromised cats (Dillon, 1998; Akuffo et al., 2018). In addition, the occurrence of animals molecularly positive for *D. immitis* and negative by all other tests may result from the residual presence in the bloodstream of dead nematode fragments. Overall, diagnosis of heartworm infection and disease in cats may be hindered by the lack of detectable antigens, antibody cross-reactivity within the genus *Dirofilaria*, and/or the presence of dead worms, suggesting that thoracic radiography and echocardiography should be used to confirm or exclude *D. immitis* infections in cats (Litster and Atwell, 2008; Hoch and Strickland, 2008). Given that cat samples here analyzed were retrospectively selected from a previous study, data on imaging investigations are missing, and no heat pre-treatment procedure was performed to exclude immune complex formation (Beall et al., 2017), which represents a limitation of the current study.

Phylogenetic analyses showed a close relationship between *D. immitis* sequences herein detected from Italy and those retrieved from dogs living in the Mediterranean Basin, suggesting that the same isolates circulate in these areas regardless of host species and geographical origin. In addition, the close phylogenetic relationship between *D. repens* sequences from cats and those from humans, may suggest the role of cats in the *D. repens* zoonotic transmission cycle.

Although hyporexia and dandruff were statistically associated with *Dirofilaria* spp. infection in cats, these are non-specific clinical findings, often associated with other chronic inflammatory conditions or infectious diseases including feline leishmaniosis (Pennisi et al. 2020). On the other hand, dyspnoea was the predominant symptom and statistically associated with *Dirofilaria* spp. infection, being regarded as the most common finding in sick cats with cardiopulmonary dirofilariosis (Litster and Atwell, 2008); however, due to the low number of positive cats retrieved and the absence of imaging diagnosis results, the above data need further confirmation. Specifically, as suggested by the presence of animals coinfect ed by *D. immitis* and *L. infantum*, clinical signs and clinicopathological abnormalities herein reported should be interpreted carefully, considering the possible occurrence of other concomitant diseases and/or co-infections that were not ruled out in the studied feline population.

In summary, this study provides data on the current epidemiological status of *Dirofilaria* spp. infections in cats from different countries of the Mediterranean Basin, suggesting that *D. immitis* mainly circulates in cats from islands such as Ibiza (Spain) and Kythnos (Greece). In addition, considering the lack of consensus between diagnostic tests herein employed, as well as the non-specific clinical presentation and laboratory abnormalities of infected cats, a multimodal approach for *Dirofilaria* infection detection is advocated. Under the circumstances of strategic chemoprophylaxis treatments of dog populations, monthly administration of drugs with proven chemoprophylactic effect, such as selamectin, moxidectin, ivermectin and milbemycin oxime, are recommended, in different associations and formulations, also for cats living in endemic areas, being a safe and effective option to prevent infection in the feline host, especially considering the challenges in diagnosis and therapy for this animal species.

### CRediT authorship contribution statement

**Mariaelisa Carbonara:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Livia Perles:** Writing – review & editing, Methodology, Formal analysis, Data curation. **Luigi Venco:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Simona Gabrielli:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Vanessa R. Barrs:** Writing – review & editing, Methodology. **Guadalupe Miró:** Writing – review & editing, Methodology, Investigation. **Elias Papadopoulos:** Writing – review & editing, Methodology, Investigation. **Clara Lima:** Writing – review & editing, Methodology, Investigation. **Emilie Bouhsira:** Writing – review & editing, Methodology, Investigation. **Gad Baneth:** Writing – review & editing, Methodology, Investigation. **Nikola Pantchev:** Writing – review & editing, Methodology, Investigation. **Roberta Iatta:** Writing – review & editing, Methodology, Investigation. **Jairo Alfonso Mendoza-Roldan:** Writing – review & editing, Methodology, Investigation. **Nicola Decaro:** Writing – review & editing, Methodology. **Bettina Schunack:** Writing – review & editing, Methodology. **Giovanni Benelli:** Writing – review & editing, Methodology, Formal analysis, Data curation. **Domenico Otranto:** Writing – review & editing, Writing – original
draft, Supervision, Investigation, Formal analysis, Data curation, Conceptualization.

## Acknowledgements

The study has been partially sponsored by Elanco Animal Health and by IDEXX Laboratories. The authors would like to thank other colleagues who contributed to the collection and/or processing of samples (i.e., Dr. Yaarit Nachum-Biala from Israel; Dr. Ana Montoya from Spain; the Greek Voluntary Veterinary Action from Greece; Dr. Elena Bossolini, Dr. Renata Fagundes-Moreira, Dr. Natalizia Palazzo and Dr. Giulia Roncetti from Italy; Dr Julie Vigier and all the practitioners from France). DO, RI and JAMR were partially supported by European Union (EU) funding within the Next Generation EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT); CL by the Portuguese Foundation for Science and Technology (FCT) through the PhD scholarship 2020.07306 (DOI 10.54499/2020.07306.BD). This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.

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