103 Introduction In the Mediterranean basin, Leishmania infantum is transmitted by dipteran insects of Phlebotomus genus Phlebotomus (Larroussius) subgenus, including the main Italian vector P.  perniciosus (Busani et  al. 2012). Seroprevalence of canine leishmaniosis (CanL) varies between 10 and 37% (Solano‑Gallego et  al. 2011). Symptomatic and asymptomatic dogs are sources of the parasite and phlebotomine sand flies play an active role in the transmission of Leishmania to humans (Molina et  al. 1994). Seroprevalence in southern Europe has ranged from less than 5% to more than 50% according to the geographical areas. However, the prevalence is significantly higher than seroprevalence and may even exceed 65%, as demonstrated by the detection of specific Leishmania cellular immunity (Cabral et  al. 1998, Cardoso et al. 1998) and by the detection of parasite's DNA in seronegative dogs (Solano‑Gallego et  al. 2001, Fernández‑Bellon et  al. 2008). In fact, the majority of dog population is exposed and becomes infected without showing clinical evidence of disease or even antibodies (Baneth et al. 2008). Sicily is a region highly endemic for CanL (Brianti et  al. 2014, Brianti et  al. 2016) and approximately 47% of the Sicilian population lives in areas at risk for visceral leishmaniasis (Cascio et al. 2002), thus making an early diagnosis, coupled with vector control strategies in cat, human and dog populations, necessary. Several studies have also been conducted on Sicilian islands, such as in Lampedusa island (Foglia Manzillo et  al. 2018). This study showed that more than 50% of dogs tested was sieropositive while in Lipari and Vulcano islands, prevalence was 41.7% and 23.6%, respectively (Otranto et al. 2017). Materials and methods Study area The present study was carried out in Pantelleria, a small island (80 km2) (36°47’27”N 11°59’38”E) during February 2017. This island is located the southwest 1Istituto Zooprofilattico Sperimentale della Sicilia, Centro di Referenza Nazionale per le Leishmaniosi, via G. Marinuzzi 3, 90129 Palermo, Italy. 2Section of Dermatology, Department of Health Promotion, Maternal-Infant, Internal Medicine and Specialization of Excellence “G. D’Alessandro” (PROMISE) University of Palermo, Palermo, Italy. *Corresponding author at: Istituto Zooprofilattico Sperimentale della Sicilia, Centro di Referenza Nazionale per le Leishmaniosi, via G. Marinuzzi 3, 90129 Palermo, Italy. Tel.: +39 091 6565348, e-mail: fabrizio.vitale@izssicilia.it. Keywords Leishmania infantum, Pantelleria island, Canine leishmaniasis. Summary Dogs are the major reservoir of Leishmania infantum, the causative agent of canine visceral and cutaneous human leishmaniasis in the Mediterranean basin. Canine and human leishmaniosis are endemic in Italy, particularly in central and southern regions, including islands. Here we show a preliminary, clinical, serological and molecular study carried out in Pantelleria island during 2017. In this study, we clinically examined 136 dogs for the presence of symptoms compatible with leishmaniasis, determined the titer of anti‑Leishmania antibodies, and investigated Leishmania DNA by real time PCR in blood and/or lymph node of each dog. The prevalence of disease was equal to 27% with 95% CI [21%; 32%], lower than prevalence obtained in the other Sicily islands (Lampedusa, Lipari). We observed that enlarged lymph nodes was more positivitely associated with canine leishmaniasis (CanL) than other clinical signs. The results obtained showed that in an endemic area, such as Sicily, diagnosis of CanL needs to be carried out by including an immunological, molecular clinical approach. Fabrizio Vitale1*, Federica Bruno1, Antonella Migliazzo1, Antonella Galante1, Angela Vullo1, Raffaele Graziano1, Salvatore D’Avola1, Valentina Caputo2 and Germano Castelli1 Cross-sectional survey of canine leishmaniasis in Pantelleria island in Sicily Veterinaria Italiana 2020, 56 (2), 103‑107. doi: 10.12834/VetIt.2059.10976.3 Accepted: 02.01.2020 | Available on line: 31.12.2020 104 Veterinaria Italiana 2020, 56 (2), 103‑107. doi: 10.12834/VetIt.2059.10976.3 Cross‑sectional survey of canine leishmaniasis in Sicily Vitale et al. was carried out in a LightCycler® 96 (Roche Life Science) using 1  ×  TaqMan Universal Master Mix (Applied Biosystems, Monza, Italy) and performed as previously described (Vitale et al. 2004). Statistical analysis A descriptive analysis of the data was performed according to 11 variables that have been collected to evaluate possible associations with the presence of L.  infantum in dogs. Data is showed in Table I. Variables were analyzed in the logistic regression model using the STATA 9.2 software (StataCorp LP, College Station, Texas). Results Characteristics of the dog study population in Pantelleria island were summarized in Table II: 63.24% were owners dog (36.76% owned by the kennel); 58.96% of the dogs examined were males, of mixed breed (70.58%); 58.65% of the dogs in the sample were aged less than or equal to 5 years, while 71.97% of the dogs lived with one or more dogs and 89.71% underwent regular repellent tools (collars, spot‑on and spray formulations). The results of the evaluation of typical clinical signs of CanL were expressed in percentages (%) and are shown in Table III. Clinically, 30/136 (25.73%) of the animals showed at least one of Sicily and 60 km (37 miles) east away from the Tunisian coast. Dogs largely represent the most abundant domestic animals present on the island; less than 1,000 dogs have been estimated. Sampling The population study comprised 136 stray and domestic dogs. The dogs were selected based on the owners’ willingness to have their pet included in the survey. Dogs were of different sex, breed and age. Clinical assessment was performed and all data was recorded including signaling, anamnestic history, repellents used and clinical examination. Finally, a dermatologic examination for ectoparasites and changes compatible with canine leishmaniosis (e.g., alopecic, nodular, ulcerative, crusty, or scaly dermatitis) was conducted. For each dog a blood sample was performed, while from 102/136 dogs needle aspirates of lymph nodes were collected. All samples (serum, whole blood, lymph node aspirate) were sent to the National Reference Centre for Leishmaniosis (C.Re.Na.L.). Anti‑Leishmania antibody detection by IFAT The collected serum samples were tested by IFAT for the L.  infantum antibodies (Bio Merieux Spa, Florence, Italy). DNA extraction and real time PCR assays Total DNA was extracted from EDTA blood and lymph node aspirate using an E.Z.N.A Tissue DNA kit (Omega biotech VWR, Norcross, GA, USA) following the manufacturer’s instructions. The real‑time polymerase chain reaction (RT‑PCR) Table I. Variables considered in the analysis. Variable Description and coding IFAT Serological test result (0 = negative; 1 = positive) Dog owner 0 = Domestic dog; 1 = Kennel Sex 0 = Male; 1 = Female Age ≤ 5 years; > 5 years Dog breed 0 = Purebreed dog; 1 = mixed-breed dog Repellent tools 0 = never/ occasionally; 1 = Regularly Cohabitation with other dogs 0 = No; 1 = 1 or more Slimming 0 = No; 1 = Yes Skin signs 0 = No; 1 = Yes Enlarged lymph nodes 0 = No; 1 = Yes Ocular signs 0 = No; 1 = Yes Table II.Characteristics of the canine population in Pantelleria and variables considered in the study, absolute frequencies and percentage values. Absolute frequency % Dog Dog owners 86 63.24 Kennel 50 36.76 Sex Male 79 58.96 Female 55 41.04 Breed Purebred dog 40 29.42 Mixed breed 96 70.58 Age ≤ 5 years 78 58.65 > 5 years 55 41.35 Repellent tools Never/ occasionally 14 10.29 Regularly 122 89.71 Cohabitation with other dogs Yes 95 71.97 No 37 28.03 105Veterinaria Italiana 2020, 56 (2), 103‑107. doi: 10.12834/VetIt.2059.10976.3 Vitale et al. Cross‑sectional survey of canine leishmaniasis in Sicily mL) than lymph node. Out of 29 dogs qPCR positive, 13 showed no symptoms of leishmaniasis (weight loss, skin and ocular signs and enlarged lymph nodes); the remaining 16 dogs showed one or more symptoms mentioned above. The prevalence of the infection was 27% (95% CI, 21%‑32%). Discussion CanL constitutes a considerable veterinary challenge, as well as an important public health problem. Pantelleria island (Sicily) is featured by optimal conditions to study a well‑defined population of animals and pathogens. The prevalence of Leishmania infection, estimated from the model, was 27% (95% CI, 21%‑32%). This prevalence was lower than Lipari island (41.7%) but similar to the Vulcano island (23.6%) (Otranto et al. 2017), while seroprevalence (30.88%) was lower than Lampedusa island seroprevalence (54%) (Foglia Manzillo et al. 2018). This variability in canine seroprevalence in Sicilian islands could be due to differences in disease surveillance and, importantly, for the presence of a kennel in Pantelleria which surely promotes stray dog population control. This finding, however, could be also related to the differences of the population under examination. Repellent tools (collars, spot‑on and spray formulations) performed on 90% of the dog study population had surely a protective effect. Repellent typical clinical sign of CanL: 6.62% of dogs showed weight loss, 11.85% cutaneous signs, 3.73% ocular signs while 16.18% of examined dogs had enlarged lymph nodes 30.88% of the dogs examined (42/136) were positive at IFAT with titers ranging from 1:160 to 1:5,120 and 51 of 136 exhibiting an antibody titer from 1:40 to 1:80. Table IV shows the results obtained in the logistic regression after applying the stepwise backward variable selection method. The results obtained from the analysis showed that the age and the enlarged lymph nodes were positively associated with serum‑positivity to IFAT, ie dogs over 5 years of age had a probability of being positive with L.  infantum 3 times higher compared to older dogs less than or equal to 5 years; while dogs with enlarged lymph nodes were 4 times more likely to be seropositive to L.  infantum than dogs with no enlarged lymph nodes. Real time PCR assay (qPCR) was carried out from 136 blood and 102 lymph node samples, providing useful information to support the tissue of choice for diagnosis of CanL. Among qPCR 73/102 negative dogs 27 samples showed negative IFAT titre (< 1:40), 24 displayed a threshold titre (from 1:40 to 1:80) and 22 dogs were positive (≥ 1:160) (Table  V). Twenty‑nine dogs (28.43%) tested positive for L. infantum DNA by qPCR from lymph node matrices with different parasite load. Only one dog tested qPCR positive (0.73%) in blood showing higher parasite load (> 1000 Leishmania/ Table III. Clinical signs in absolute frequencies and percentage values observed in dog. Absolute frequency % Clinical signs Yes 30 25.73 No 106 74.27 Weight loss Yes 9 6.62 No 127 93.38 Cutaneous signs Yes 16 11.85 No 119 88.05 Enlarged lymph nodes Yes 22 16.18 No 114 83.82 Ocular signs Yes 5 3.73 No 126 96.27 Cohabitation with other dogs Yes 95 71.97 No 37 28.03 Table IV. Coefficient and OR for logistic regression on IFAT and age, enlarged lymph nodes. Variables Coefficient P-value OR 95% CI for OR Lower Upper Age 0.981 0.017 2.67 1.192 5.961 Enlarged lymph nodes 1.357 0.017 3.89 1.277 11.825 CI = Confidence interval; OR = Odds ratio. Table V. Detection and accurate parasite quantification of the qPCR in lymph nodes aspirates. Lymph node aspirates qPCR Negative IFAT titre (< 1:40) Threshold IFAT titre (1:40 and 1:80) Positive IFAT titre (≥ 1:160) Total Negative 27 24 22 73 1-10 Leishmania/ml 0 2 4 6 10-100 Leishmania/ml 0 3 2 5 100-1,000 Leishmania/ml 1 4 2 7 > 1,000 Leishmania/ml 0 2 9 11 102 106 Veterinaria Italiana 2020, 56 (2), 103‑107. doi: 10.12834/VetIt.2059.10976.3 Cross‑sectional survey of canine leishmaniasis in Sicily Vitale et al. one dog showed parasite's DNA in blood, this study also suggests that blood matrices is inadequate for qPCR, since hematogenous dissemination occurs only rarely. On the other hand, these results indicated that lymph nodes were the matrix of choice for molecular detection of CanL in dogs (Martínez et al. 2011, Moreira et al. 2007). Overall, the combination of serology, direct detecton of DNA and clinical examination should be performed in order to reach a correct diagnosis. The absence of reports of clinical case of human cutaneous/visceral leishmaniasis infection in Pantelleria island during the period of our study could be related to asymptomatic Leishmania infections in immunocompetent hosts without clinically evident disease. Acknowledgements This research was granted by Ministry of Health RC IZS SI 01/2017. tools remains the most effective antivectorial method to control L.  infantum. Furthermore, the presence of seropositivity of dogs older than 5 years (30.88%), was positively associated with Leishmania infection. During investigation, cutaneous and ocular signs and weight loss observed were related to Leishmania disease in dogs, since percentages of association obtained are very low. Only enlarged lymph nodes were positively associated with Leishmania infection, even if only 16,18 % of dogs presented this clinical sign (Table IV). Early CanL diagnosis was crucial to identify infectious dogs through several diagnostic tests, but the correct interpretations of these were of great importance to make an accurate diagnosis of the disease. IFAT test ‘gold standard’ technique for mass screening dogs, could however generate false positive results due to both serological cross‑reactivity with other pathogens and low sensitivity in asymptomatic dogs. The use of qPCR for the assessment of parasite load could be more informative. However, as only 107Veterinaria Italiana 2020, 56 (2), 103‑107. doi: 10.12834/VetIt.2059.10976.3 Vitale et al. 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