379 Please refer to the forthcoming article as: Vilcek et al. 2022. High prevalence of Leishmania spp. in canines from Central West Colombia. Vet Ital. 10.12834/VetIt.2632.16820.2. are pathogenic for humans (Akhoundi et al., 2016). These species are grouped by complexes according to the tropism to a specific tissue (Akhoundi et al., 2016) and they can infect macrophages in the bone marrow, lymph nodes, spleen, liver, kidneys, gastrointestinal tract, skin, and mucous membranes (Reis et al., 2009). This leads to the presentation of three clinical forms: cutaneous, mucocutaneous, and visceral leishmaniasis, characterized by presenting macroscopic lesions such as lymph node hypertrophy, ulcerative dermatitis (periorbital, nasal Introduction Leishmaniasis is a zoonotic disease caused by the invasion of protozoan parasites of the Leishmania genus, transmitted to mammals mainly by the hematophagous activity of sandflies belonging to the Lutzomyia and Phlebotomus genera (Ribeiro et al., 2018; Torres et al., 2017). This disease has a worldwide incidence of 0.7 million new cases per year and up to 65,000 deaths are reported annually (Alvar et al., 2012). There are around 53 species of Leishmania, of which parasitize mammals and 20 Veterinaria Italiana 2022, 58 (4), 379‑389. doi: 10.12834/VetIt.2632.16820.2 Accepted: 07.01.2022 | Available on line: 31.12.2022 1Research Group in Immunobiology and Pathogenesis, Faculty of Veterinary Medicine and Zootechnics, University of Tolima, Ibagué, Colombia. 2 Municipal Health Secretary, Mayor of Ibagué, Colombia. *Corresponding author at: Research Group in Immunobiology and Pathogenesis, Faculty of Veterinary Medicine and Zootechnics, University of Tolima, Ibagué, Colombia. E‑mail: lagiraldom@ut.edu.co. Leidy Alejandra Giraldo‑Martínez 1*, Julieth Michel Petano‑Duque1, Heinner Fabian Uribe‑García1, Roberto Andrés Chacón‑Novoa1, Blanca Lisseth Guzmán2, Iang Rondón‑Barragán1 Keywords Canine leishmaniasis, Canine, Hsp70, ITS‑, Risk factors. Summary Leishmaniasis is a widespread disease caused by species of the genus Leishmania. In Colombia, this zoonosis is endemic in rural areas with a high prevalence in the departments of Antioquia, Santander, Meta, Tolima and Nariño. Dogs are the most important domestic reservoirs of the pathogen, given the epidemiological importance of dogs in the control of leishmaniasis is needed to determine the prevalence of Leishmania spp. in canines of the rural area of Ibagué and to identify potential risk factors related to the presence of this parasite. A cross‑sectional study was carried out in 173 dogs from the rural area of Ibagué. Leishmania spp. was detected by amplifying the Internal Transcribed Spacer (ITS‑1) and two regions of the hsp70 gene through PCR. Factor associations were calculated through the Chi‑square and odds ratio. Prevalence of Leishmania spp. infection in dogs was of 91.33% (158/173), where 36.71% (58/158) of the Leishmania spp. positive dogs showed one or more clinical signs of canine leishmaniasis and 63.29% (100/158) of the dogs were asymptomatic. Factors associated with the presence of the parasite did not show significant association. In addition, hsp70D‑PCR was proved to be highly efficient for the detection of Leishmania spp. High prevalence of Leishmania spp. in from Central West Colombia Leishmania spp. in dogs, Central West Colombia Leidy Alejandra Giraldo-Martínez et al. 380 Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 has a population of 569,336 inhabitants (Centro de información municipal para planeación participativa, 2021) and a canine population of 40,016 animals (Ministerio de Salud y Protección Social de Colombia, 2018). Rural area is organized into 17 townships with approximately 144 villages. Sample size Sample size was estimated based on a population of 40,016 canines in the 17 townships of the rural area of Ibagué (Ministerio de Salud y Protección Social de Colombia, 2018) using the formula described by Thrusfield (2018), where the expected prevalence was 10%, the level of significance was 95% and the desired absolute precision was 5%. Clinical examination, epidemiological survey and blood sampling Study was carried out between July and November of 2019. Each canine`s owner signed a consent form before any procedure with the animals. Clinical examination was performed by a physical inspection to detect signs compatible with leishmaniasis including dermatological lesions (ulcers, alopecia, erythematous lesions), onychogryphosis, apathy, low body condition and abnormal peripheral lymph nodes. A survey was fulfilled in order to find possible risk factors related to the presence of Leishmania spp. in the canines, data included canine breed, dwelling features (a type of material of the house and the floor) and dwelling surround characteristics (disposal of garbage, presence of other animals, forest areas, agricultural activities and streams near the home). One hundred seventy‑ three samples of canine blood were collected by peripheral venipuncture in the saphenous or cephalic vein. Samples were placed in tubes with ethylenediaminetetraacetic acid and transported in an ice box until storage at ‑20 °C. DNA extraction and endpoint PCR Total genomic DNA extraction was performed from blood samples using phenol‑chloroform‑isoamyl alcohol (25:24:1). DNA quality was verified by NanoDrop One spectrophotometer (ThermoFisher, USA) and stored at ‑20 °C until its use (Wang et al., 2011). Leishmania spp. was detected by amplifying the Internal Transcribed Spacer 1 (ITS1) and regions C (1545‑1778) and D (711‑1089) of the hsp70 gene (Table 1). Animals were considered positive to Leishmania spp. if one of the three tests was positive. PCR was performed in a T‑100 thermal cycler (Bio‑Rad, or disseminated), and edema in the limbs (Mümtaz, 2018). Leishmaniasis has a high prevalence in tropical developing countries and mainly affects the population in low‑income countries (Oryan & Akbari, 2016). In Colombia, it is distributed in rural areas, with high prevalence in the departments of Antioquia, Santander, Meta, Tolima, and Nariño (Mendigaña, 2019), where nine species have been reported: Leishmania panamensis, L. braziliensis, L. guyanensis, L. infantum, L. colombiensis, L. amazonensis, L. equatoriensis, L. lansoni and L. mexicana (Salgado‑ Almario et al., 2019; Ramírez et al., 2016). According to the 2019 epidemiological bulletin were reported in the department of Tolima, 13 belonging to the municipality of Ibagué (incidence rate of 2.26) (Sistema de Vigilancia en Salud Pública, 2019). In 2020, 32 cases of CL have been notified with an incidence rate of 5.52% (Macedo & Urrego, 2020). Infection with Leishmania spp. in canines can be clinical or subclinical, becoming a public health concern due to the risk of transmission to humans in the presence of hematophagous vectors (Chinchilla & Vanessa, 2010; Flórez et al., 2006; Zoghlami et al., 2014). Several risk factors have been described related to Leishmania spp. infection including canine breed, infestation with ticks, proximity or contact with other animals (e.g. chickens, pigs, horses), improper garbage disposal, dwelling proximity to water sources, forest areas, and informal crops (Membrive et al., 2012; Oryan & Akbari, 2016; Silva et al., 2018). A higher prevalence of visceral leishmaniasis has been reported in shorthaired canines and purebreds such as Boxer, Pit Bull Terrier, Cocker Spaniel, and Great Danish (Almeida et al., 2010; Belo et al., 2013b; França‑Silva et al., 2003). Active epidemiological surveillance based on the detection of infected animals is an initial step to establish appropriate control measures. Clinical examination allows the first approach to the diagnosis, however, in asymptomatic animals the use of diagnostic tests such as the Polymerase Chain Reaction (PCR) is required (Cavalcanti et al., 2012; Reis et al., 2013; Zoghlami et al., 2014). In the municipality of Ibagué, human leishmaniasis has been reported, nevertheless, the role of canines in the epidemiology of leishmaniasis is unknown. Thus, the present study aimed to determine the prevalence of Leishmania spp. in canines of the rural area of Ibagué and to identify potential risk factors related to the presence of this parasite. Material and methods Study location Study was carried out in Ibagué, the capital city of the department of Tolima, Colombia, which Leidy Alejandra Giraldo-Martínez et al. Leishmania spp. in dogs, Central West Colombia Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 381 hsp70 gene of Leishmania spp. were submitted to GenBank in order to obtain accession numbers (MW509745, MW509746, MW509747, MW509748 and MW509749). Statistical analysis A database was created from the information collected in the clinical examinations and the epidemiological survey in the Epi Info software (CDC, Atlanta, Georgia, USA). Analysis of the independent variables in the epidemiological survey was performed using GraphPad Prism v.8 for macOS (La Jolla, CA, USA). Frequency tables were constructed and their relationship with the presence of Leishmania spp. was established through the Chi‑square. Strength of this relationship was determined by calculating the odds ratio. Results Characteristics of the canine population A total of 173 dogs were sampled. They were distribuited 65 from Totumo, 32 from Coello cocora, 14 from El Salado, 13 from Cay, 11 from San Bernardo, 11 from Calambeo, 9 from Gamboa and 9 from Carmen de Bulira, 9 from Buenos Aires. Of the 173 sampled dogs, 57.8% were males and 42.2% females. Crossbreed dogs were 79.19% and purebred 20.81%, they had short fur (65.32%) with an average age of 48.9 months. All animals were dogs with an owner, but 31.8% did not stay in the dwelling during the day and 22.5% did not stay overnight. Regarding the clinical sign compatible with leishmaniasis, 10.4% of the dogs had foci of alopecia, 12.72% skin lesions, 24.85% pruritus, and 2.89% onychogryphosis and lymphadenitis (Figure 1). Table I. Sequences of primers for amplification of ITS1 and hsp70 genes. Gene Amplicon size (bp) Primer sequence (5´-3´) Reference ITS1 314 F: CTGGATCATTTTCCGATG Schönian et al. (2003)R: TGATACCACTTATCGCACTT hsp70C 234 F: GGACGAGATCGAGCGCATGGT Graça et al. (2012)R: TCCTTCGACGCCTCCTGGTTG hsp70D 379 F: CCGCCTCGTCACGTTCTTCAGC Graça et al. (2012)R: GTTCAGCTCCTTGCCGCCGA Hercules, California, USA) using a final reaction volume of 25 μL, composed of 14.875 μL of distilled and deionized water (ddw), 5 μL of 5x Colorless GoTaq Flexi Buffer, 1 μL of dNTPs at 1.5 mM, 1 μL of each primer (forward and reverse) [10 pmol/μL], 1 μL MgCl2 at 25 mM, 0.125 μL of GoTaq Flexi DNA polymerase (Promega, Madison, Wisconsin, USA) and 1 μL of the gDNA sample as template. Amplification steps consisted of initial denaturation cycle at 95 °C for 3 minutes, followed by 35 denaturation cycles at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 30 seconds, and final extension at 72 °C for 5 minutes. Positive control of L. infantum (MCAN/ CN/90/SC) and negative control (ddw) were used. PCR products were revealed by 2% agarose gel electrophoresis, stained with Hydragreen (ACTGene, Piscataway, New Jersey, USA), for 40 minutes at 100 V in the myGel Mini electrophoresis chamber (ACCURIS, Edison, New Jersey, USA) and visualized under ultraviolet light using an ENDURO GDS (LabNet Intl, Edison, New Jersey, USA). DNA sequencing and phylogenetic analysis hsp70C‑PCR products of 234 pb were sequenced by the Sanger method (Macrogen, Seoul, Korea), sequencing was carried out by the same primer pair (hsp70C). Consensus sequences from forward and reverse reads were edited and analyzed using Geneious Prime software (Version 2021.0.3) (Biomatters, Auckland, New Zealand) (Kearse et al., 2012), furthermore they were compared with the GenBank database (NCBI, USA) by using the BLASTN tool (Version 2.2.28+). Bioinformatic analysis included multiple alignments of our sequences and 18 hsp70 sequences from 7 Leishmania species reported in GenBank database (NCBI, USA), and the construction of phylogenetic trees using Neighbor‑Joining (NJ) method, with a bootstrap consensus inferred from 1000 replicates. It was used as outgroup the region C of hsp70 gene sequence of Trypanosoma cruzi (GenBank accession No. MF144929.1). Sequences of the region C of Figure 1. Dogs with cutaneous lesions, mucocutaneous lesion, localized alopecia, cutaneous hypopigmentation and onychogryphosis hypopigmentation and onychogryphosis. Leishmania spp. in dogs, Central West Colombia Leidy Alejandra Giraldo-Martínez et al. 382 Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 the peridomiciliary zone, 56.65% of the ares had a water stream of 56.65%, wooded areas, and in 71.68% there was presence of mosquitoes. Of the dog's owners, 67.63% used protective measures for the mosquitoes, like bed nets to sleep (66.96%), and the remaining used repellents or fumigated their household (33.04%). According to the statistical analysis, characteristics of the canine, sociodemographic factors, home conditions and social interactions evaluated did not show associations with the presence of Leishmania spp. in the canines of Ibagué, and therefore, statistically significant risk factors were not determined. Phylogenetic analyses PCR products of hsp70C region (234 bp) from the LE_019, LE_021, LE_022, LE_024, LE_025, LE_029, LE_030, LE_053 and LE_064 samples were sequenced. Sequences LE_019 and LE_029 LE_025 have 100% identity with each other, the same as LE_021, LE_053 and LE_064, therefore, 5 partial sequences of the hsp70 gene of Leishmania spp. were submitted to GenBank. Accession numbers of sequences were as follows: LE_019 (MW509745), LE_021 (MW509746), LE_022 (MW509747), LE_024 (MW509748) and LE_030 (MW509749). Phylogenetic analysis bootstrapping provided strong support for all the nodes. Leishmania subgenres were separated for one cluster supported by 99.3%. Viannia subgenus, this was divided in L. braziliensis complex species and L. equatorensis with a support of 61.5%. Leishmania subgenus was divided in L. mexicana complex and L. donovani complex, where sequences of this study cluster with L. donovani complex (Figure 2). Molecular diagnosis Leishmania spp. were detected by amplification of 3 different targets by PCR assay, hsp70C, hsp70D and the ITS1 of the parasite. One hundred fifty eight of the 173 dogs were positive for Leishmania spp. in at least one of the three tests, which represents a prevalence of 91.33%. On the other hand, 63.29% (100/158) of the PCR‑positive dogs did not show any clinical sign compatible with the disease and only 36.71% (58/158) of the dogs presented one or more clinical signs of canine leishmaniasis (Table 2). In addition, 4.43% (7/158) of the positive dogs remain during the day in the houses, and 79.75% (126/158) stayed outside of the house at night, in the backyard or in a close neighborhood. Efficiency of different PCR assays was compared, taking as a parameter the number of samples detected as positive for the presence of the parasite. One hundred thirty‑eight DNA samples were positive for the hsp70 gene of Leishmania spp. through the hsp70C‑PCR, 140 by using the hsp70D‑ PCR, and 22 DNA samples were positive for the ITS‑1 of Leishmania spp. (Table 2). According to which the hsp70D PCR was proven to be highly efficient for the detection of Leishmania spp. and ITS1‑PCR had the fewest detections of the parasite in canine blood. Risk factors Regarding the housing conditions of the dogs, the wall and floor materials were mostly concrete (65.32% and 64.16%, respectively), the garbage disposal more frequent was in the municipal landfill (41.04%) and burning (35.26%), the 49.71% had agricultural production, specifically poultry. In Table II. Distribution of Leishmania-positive canines by village. Village Detection of Leishmania spp. Clinical signs ITS1 hsp70D hsp70C Positive canines (n) + - + - + - + - Buenos Aires 0 9 9 0 9 0 9 6 3 Calambeo 0 11 11 0 11 0 11 4 7 Carmen de Bulira 4 5 6 3 6 3 9 4 5 Cay 0 13 13 0 13 0 13 9 4 Coello Cocora 5 27 26 6 26 6 28 9 19 El Salado 2 12 5 9 4 10 12 5 7 Gamboa 6 3 6 3 2 7 9 2 7 San Bernardo 0 11 11 0 11 0 11 0 11 Totumo 5 60 53 12 56 9 56 19 37 Total 22 151 140 33 138 35 158 58 100 Leidy Alejandra Giraldo-Martínez et al. Leishmania spp. in dogs, Central West Colombia Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 383 Solano‑Gallego et al., 2001). In Brazil, prevalence is reported between 3.2% to 50.3%, depending on the area and methods (Marcondes & Day, 2019), in this endemic country, serological tests were found to have low capacity for Leishmania spp. detection when it is compared with molecular tests in canines (Lopes et al., 2017). The most widely methods used for the diagnosis of canine leishmaniasis in Latin America are the ELISA, IFAT and Western blot technique as well as PCR (Alves & Bevilacqua, 2004; Trevisan et al., 2015). PCR has high sensitivity and specificity, and its use reduces the costs of the Leishmania spp. detection (de Paiva et al., 2009; Sundar & Rai, 2002). On the other hand, PCR can detect the presence of Leishmania spp. in Discussion Molecular diagnosis Prevalence of Leishmania spp. in canine population in Ibagué (91.33%) is highest compared with that reported by molecular methods in Chaparral‑ Tolima (7.3%) (Santaella et al., 2011), Sincelejo‑Sucre (34.9%), Sampúes‑Sucre (35.7%) and Ovejas‑Sucre (11.1%) (Paternina‑Gómez et al., 2013). In other countries, prevalence values were 24.8% by qPCR in Sichuan Province‑China, 48.4% by IFAT in Italy, 26% by ELISA and 63% by PCR in Mallorca‑Spain, and 66% by immunoblot and 80% by PCR in France (Berrahal et al., 1996; Paradies et al., 2006; Shang et al., 2011; Figure 2. Phylogenetic tree of Leishmania species and the studied samples, based in hsp70C region sequence. A bootstrapped NJ tree constructed using Geneious Prime software (Version 2021.0.3). Numbers at nodes represent the percentage of 1000 bootstrap iterations supporting the branch. T. cruzi was used as outgroup. Leishmania spp. in dogs, Central West Colombia Leidy Alejandra Giraldo-Martínez et al. 384 Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 different types of clinical samples such as blood, lymph nodes, skin, conjunctival smear and bone marrow aspirates (Lachaud et al., 2002; Lombardo et al., 2012; Manna et al., 2004; Reale et al., 1999; Reis et al., 2013). Several studies reported that amplification of ITS1 through PCR is highly specific for detection of Leishmania spp. and highly sensitive as it is capable of detecting 0.2 parasites per sample (Schönian et al., 2003; Toz et al., 2013). Graça et al. (2012) reported a sensitivity of 81.4% of ITS1‑PCR and 73.2% of hsp70C‑PCR, using DNA extracted from skin biopsies. However, in this study, sensitivity of ITS1‑ PCR is lower than that obtained with hsp70‑PCR, this may be due to the fact that the blood is less specific (80%) and sensitive (85%) than the aspirates of the lymph nodes (specificity, 100%; sensitivity, 100%) (Reale et al., 1999), given that blood tends to have a variable parasite load depending on the stage of infection (Manna et al., 2004). Nonetheless, the use of blood for the diagnosis of Leishmania spp. has shown to be effective in the detection of the parasite mainly in asymptomatic animals (Monteiro et al., 2019)” for “… PCR is highly specific for detection of Leishmania spp. and highly sensitive as it may detect 0.2 parasites per sample … sensitivity of ITS1‑ PCR is lower than that obtained with hsp70‑PCR, this may be due to the different matrix (Reale et al., 1999; Manna et al., 2004).Nonetheless, use of blood for the diagnosis of Leishmania spp. has shown to be effective in the detection of the parasite mainly in asymptomatic animals (Monteiro et al., 2019) as in our study and it is recommended as more suitable compared with aspiration‑based methods (Albuquerque et al., 2017). Furthermore, blood sampling is a procedure,simple to perform, less stress, with low cost and its repeatability is easier compared to bone marrow sampling or biopsy of lymph nodes (Carvalho et al., 2009; Lachaud et al., 2002; Reale et al., 1999). Amplification of the hsp70 gene represents a valuable tool in the detection of Leishmania spp., since it contains between five and six hsp70‑I copies followed by one hsp70‑II copy (Ramirez et al., 2011) and its effectiveness in the detection of asymptomatic infections was evidenced in the present study. hsp70 gene region is the most highly conserved in sequence and function in all organisms, with a lower rate of genetic diversity than other markers for instance gp63 gene, rDNA genes, or ITS1 (Dabirzadeh et al., 2016), becoming a relevant target for the detection of Leishmania species. Risk factors Differences in clinical manifestations between dogs, such as the severity of clinical signs and the time of onset of the disease, may vary depending on the individual immune response of the infected animals as well as genetic factors, age, nutritional status, and the virulence of Leishmania strains (Alvar et al., 2004; Man et al., 2020; Moreno et al., 1999; Quinnell et al., 2003). In our study, 63.29% of the PCR‑positive dogs did not present clinical signs of canine leishmaniasis or other symptoms. Similarly, it has reported that in endemic areas of visceral leishmaniasis 85% of dogs infected by L. infantum are asymptomatic (Dantas‑ Torres & Brandao‑Filho., 2006). Nonetheless, it has been described that approximately 46% of infected dogs acquire the infection and develop the disease immediately, another 44% of dogs develop the disease later, and 10% of them can remain without clinical signs of visceral leishmaniasis throughout their lives (Moreno & Alvar, 2002). Dogs have the ability to transmit Leishmania spp. to the vector and, consequently, to other canines or humans regardless of whether they present or no clinical signs. The transmission cycle of the parasite could be favored by the asymptomatic infection of Leishmania spp. in the canine (Dantas‑Torres & Brandao‑Filho, 2006; Moshfe et al., 2009; Soares et al., 2011). Presence of Lutzomyia longiflocosa has been reported in the rural zone of Ibagué, Tolima (Guzmán‑Barragán et al., 2021). Furthermore, in several Tolima municipalities (Planadas, Rovira, Casablanca, Herveo, Ortega, San Antonio and Chaparral), it has been reported the presence of L. columbiana, L. longiflocosa, L. micropyg, L. rangelina, L. suapensis, L. nuneztovari, L. atroclatava (Bejarano et al., 2003; Bejarano et al., 2006; Cárdenas et al., 1999; Contreras et al., 2012; Morales et al., 1981; Pardo et al., 2006; Prado et al., 1999; Sierra et al., 2000). Transmission potential of infected dogs can differ between symptomatic and asymptomatic animals. By xenodiagnosis methods it has been observed that asymptomatic dogs have a lower capacity to infect sandfly vectors (Travi et al., 2001; Verçosa et al., 2008), while other studies determined that symptomatic animals are more likely to spread the infection (Guarga et al., 2000; Molina et al., 1994). Some studies have determined that the physical traits of dogs such as height, spaying, purebred, and age (older than 2 years) can have a positive correlation with the occurrence of leishmaniasis (Belo et al., 2013a). Additionally, associated factors such as the presence of ectoparasites, contact with other animals, dog’s permanence in the backyard, forest areas near dwelling and the presence of birds in the domestic environment, may play a role in attracting sandflies and predispose the infection of dogs (Belo et al., 2013b; Curi et al., 2014; Coura‑Vital et al., 2011). However, significant associations were Leidy Alejandra Giraldo-Martínez et al. Leishmania spp. in dogs, Central West Colombia Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 385 Conflict of Interest Statement The authors declare no conflict of interest. Statement of animal rights All the experimental procedures followed the Guidelines of the Bioethics Committee of the Central Research Office of the University of Tolima based on Law 84/1989 and Resolution 8430/1993 and complied with the guidelines for animal care and use in research and teaching. The Bioethic committee of the Central Research Office approved bioethics aspects in the agreement 2239 of June 25 at 2019, between the Municipal Health Secretariat and the University of Tolima. References Akhoundi M., Kuhls K., Cannet A., Votýpka J., Marty P., Delaunay P. & Sereno D. 2016. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis, 10(3), e0004349. https://doi. org/10.1371/journal.pntd.0004349 Albuquerque A., Campino L. & Cardoso L. 2017. Evaluation of four molecular methods to detect Leishmania infection in dogs. Parasit Vectors. 10, 57. https://doi.org/10.1186/s13071‑017‑2002‑2 Almeida A., Mendonça A. & Sousa V. 2010. Prevalência e epidemiologia da leishmaniose visceral em cães e humanos, na cidade de Cuiabá, Mato Grosso, Brasil. Cienc Rural, 40, 1610‑1615. https://doi. org/10.1590/S0103‑84782010005000102 Alvar J., Cañavate C., Molina R., Moreno J. & Nieto J. 2004. Canine leishmaniasis. Adv Parasitol, 57, 1‑88. https://doi.org/10.1016/S0065‑308X(04)57001‑X Alvar J., Vélez I.D., Bern C., Herrero M., Desjeux P., Cano J., Jannin J., den Boer M. & WHO Leishmaniasis Control Team. 2012. Leishmaniasis worldwide and global estimates of its incidence. PloS One, 7(5), e35671. https://doi.org/10.1371/journal. pone.0035671 Alves W.A. & Bevilacqua P.D. 2004. Reflexões sobre a qualidade do diagnóstico da leishmaniose visceral canina em inquéritos epidemiológicos: o caso da epidemia de Belo Horizonte, Minas Gerais, Brasil, 1993‑1997. Cad Saude Publica, 20(1), 259‑265. https://doi.org/10.1590/s0102‑ 311x2004000100043 Bejarano E.E. 2006. Lista actualizada de los psicódidos (Diptera: psychodidae) de Colombia. Folia Entomológica Mexicana, 45(1),47‑56. ISSN: 0430‑8603. Bejarano E.E., Sierra D. & Vélez I.D. 2003. Novedades en la distribución geográfica del grupo not found between the risk factors evaluated and the presence of Leishmania spp. in the dogs of the municipality. This can be attributed to the immune status of the individual dogs and because of both Leishmania‑positive and Leishmania‑negative dogs have homogeneous factors, therefore odds ratios did not show significance. Additionally, other studies have shown that there are not significant associations between age and the presence of Leishmania spp. in the canine population, age is not a strong predictor for Leishmania spp. infection and dogs of all ages may be reservoirs in the study areas. Moreover, according to Belo et al. (2013b), free‑roaming dogs are more difficult targets to vector than dogs living at home; and it has been shown that the presence of chickens at home is not associated as an imminent risk factor since it does not correspond to a reservoir of the parasite. Phylogenetic analyses PCR‑hsp70C amplifies a variable region of the hsp70 gene in eleven Leishmania species (Zampieri et al., 2016). However, it was not possible to differentiate the species using the sequence obtained with the amplification carried out. All sequences reported belong to the Leishmania donovani complex, according to the geographical distribution of Leishmania species in Colombia, the Leishmania species circulating in dogs from Ibagué‑Tolima are L. infantum (Salgado‑Almario et al. 2019). This is the first molecular study of canine Leishmania infection in Ibagué, an endemic region in which a high prevalence of L. donovani complex was found. Additional investigations are required to assess the infective capacity of dogs towards sandfly vectors, looking for establishment of routine screening and preventive measures for canine and human leishmaniasis. Acknowledgments Authors thank the support provided by the officials and veterinarians from the Municipal Health Secretary, especially Dr. Leidy Parra. Grant support This work was supported by the Municipal Health Secretary of Ibagué (Agreement 2239 of June 25th, 2019) with study design, and resources from the Laboratory of Immunology and Molecular Biology, belonging to the University of Tolima, where was realized the study (conceptualization, analysis and interpretation of data, validation, writing of the report and in the decision to submit the article for publication). Leishmania spp. in dogs, Central West Colombia Leidy Alejandra Giraldo-Martínez et al. 386 Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 (CORTOLIMA) 2002. Agenda ambiental del municipio de Ibagué. Coura‑Vital W., Marques M.J., Veloso V.M., Roatt B.M., de Oliveira Aguiar‑Soares R.D., Reis L.E.S., Leoncio‑Braga S., Franco‑Morais M.H., Barbosa‑ Reis A. & Carneiro M. 2011. Prevalence and factors associated with Leishmania infantum infection of dogs from an urban area of Brazil as identified by molecular methods. PLoS Negl Trop Dis, 5(8), e1291. https://doi.org/10.1371/journal. pntd.0001291 Curi N.H., Paschoal A.M., Massara R.L., Marcelino A.P., Ribeiro A.A., Passamani M., Demétrio G.R., Chiarello A.G. & Carvalho L.H. 2014. Factors Associated with the Seroprevalence of Leishmaniasis in Dogs Living around Atlantic Forest Fragments. PLoS ONE, 9(8), e104003–. https://doi.org/10.1371/ journal.pone.0104003 Dabirzadeh M., Hashemi M. & Maroufi Y. 2016. Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran. Jundishapur J Microbio, 9(6), e33498. https:// doi.org/10.5812/jjm.33498 Dantas‑Torres F. & Brandao‑Filho S. P. 2006. Visceral leishmaniasis in Brazil: Revisiting paradigms of epidemiology and control. Rev Inst Med Trop Sao Paulo, 48, 151‑156. http://dx.doi.org/10.1590/ S0036‑46652006000300007 de Paiva M., Felinto M.E., de Souza W.V., de Miranda Y. & Abath F.G. 2009. The development of a real‑time PCR assay for the quantification of Leishmania infantum DNA in canine blood. Vet J, 182(2), 356– 358. https://doi.org/10.1016/j.tvjl.2008.05.018 Flórez M., Martínez J.P., Gutiérrez R., Luna K.P., Serrano V.H., Ferro C., Angulo V.M. & Sandoval C.M. 2006. Lutzomyia longipalpis (Diptera: Psychodidae) en un foco suburbano de leishmaniosis visceral en el Cañón del Chicamocha en Santander, Colombia. Biomédica, 26(1), 109‑20. https://doi.org/10.7705/ biomedica.v26i1.1505 França‑Silva J.C., da Costa R.T., Siqueira A.M., Machado‑Coelho G.L., da Costa C.A., Mayrink W., Vieira E.P., Costa J.S., Genaro O. & Nascimento E. 2003. Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros Municipality, Minas Gerais State, Brazil. Vet Parasitol, 111(2‑3), 161–173. https://doi. org/10.1016/s0304‑4017(02)00351‑5 Graça G.C.D., Volpini A.C., Romero G.A.S., Oliveira M.P.D., Hueb M., Porrozzi R. & Cupolillo E. 2012. Development and validation of PCR‑based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species. Mem. Inst. Oswaldo Cruz 107(5), 664‑674. Guarga J.L., Lucientes J., Peribáñez M.A., Molina R., verrucarum (Diptera: Psychodidae) en Colombia. Biomédica, 23, 341‑50 Belo V.S., Struchiner C.J., Werneck G.L., Barbosa D.S., de Oliveira R.B., Neto R.G. & da Silva E.S. 2013a. A systematic review and meta‑analysis of the factors associated with Leishmania infantum infection in dogs in Brazil. Vet Parasitol, 195(1‑2), 1–13. https:// doi.org/10.1016/j.vetpar.2013.03.010 Belo V.S., Werneck G.L., Barbosa D.S., Simões T.C., Nascimento W.L., da Silva E.S. & Struchiner C.J. 2013b. Factors Associated with Visceral Leishmaniasis in the Americas: A Systematic Review and Meta‑Analysis. PLoS Negl Trop Dis, 7(4), e2182. https://doi.org/10.1371/journal. pntd.0002182 Berrahal F., Mary C., Roze, M., Berenger A., Escoffier K., Lamoroux D. & Dunan S. 1996. Canine leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am J Trop Med Hyg, 55, 273–277. https://doi.org/10.4269/ajtmh.1996.55.273 Cárdenas R., Romo G., Santamaría E., Bello F. & Ferro C. 1999. Lutzomyia longiflocosa (Diptera: Psychodidae) posible vector en el foco de leishmaniasis cutánea del municipio de Planadas, zona cafetera del Tolima. Biomédica, 19, 239‑44 Carvalho D., Oliveira T.M.F.S., Baldani C.D. & Machado R.Z. 2009. An enzyme‑linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs. Pesqui Vet Bras, 29, 120‑124. https://doi.org/10.1590/S0100‑ 736X2009000200006 Cavalcanti A., Lobo R., Cupolillo E., Bustamante F. & Porrozzi R. 2012. Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report. Parasitol Int, 61(4), 738‑740. https://doi.org/10.1016/j. parint.2012.05.002 Centro de información municipal para planeación participativa (CIMPP) 2021. Demografía: Dane 1938‑2018. http://cimpp.ibague.gov.co/ demografia/ Chinchilla E. & Vanessa M. 2010. Estudio serológico y molecular de Leishmania spp. en cánidos domésticos de dos cantones del municipio de San Ildefonso, Departamento de San Vicente, El Salvador. Doctoral dissertation, Universidad de El Salvador. Contreras M.A., Vivero R.J., Bejarano E.E., Carrillo L.M. & Vélez I.D. 2012. Nuevos registros de flebotomíneos (Diptera: Psychodidae) en el área de influencia del río Amoyá en Chaparral, Tolima. Biomédica, 32(2). https://doi.org/10.7705/ biomedica.v32i2.438 Corporación autónoma regional del Tolima Leidy Alejandra Giraldo-Martínez et al. Leishmania spp. in dogs, Central West Colombia Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 387 rid/Lists/BibliotecaDigital/RIDE/VS/PP/ET/plan‑ estrategico‑leishmaniasis‑2018‑2022.pdf Ministerio de Salud y Protección Social de Colombia (MINSALUD) 2018. Cobertura nacional de vacunación antirrábica de perros y gatos, año 2 0 1 8 . h t t p s : / / w w w. m i n s a l u d . g o v. c o / s i t e s / r i d / L i s t s / B i b l i o t e c a D i g i t a l / R I D E / V S / P P / S A / co b e r t u r a s ‑ v a c u n a c i o n ‑ a n t i r r a b i c a ‑ p e r ro s ‑ gatos‑2018.zip Molina R., Amela C., Nieto J., San‑Andrés M., González F., Castillo J.A., Lucientes J. & Alvar J. 1994. Infectivity of dogs naturally infected with Leishmania infantum to colonized Phlebotomus perniciosus. Trans R Soc Trop Med Hyg, 88(4), 491–493.https://doi.org/10.1016/0035‑9203 (94)90446‑4 Monteiro F.M., Machado A.S., Rocha‑Silva F., Assunção C.B., Graciele‑Melo C., Costa L.E., Portela A.S., Ferraz E.A., de Figueiredo M. & Caligiorne R.B. 2019. Canine visceral leishmaniasis: Detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real‑time polymerase chain reaction (rt‑PCR). Microb Pathog Jan, 126, 263‑268. https://doi.org/10.1016/j.micpath.2018.10.036. Morales A., Corredor A., Cáceres E., Ibagos A.L. & de Rodriguez C. 1981. Aislamiento de tres cepas de Leishmania a partir de Lutzomyia trapidoi en Colombia. Biomédica, 1, 198‑206. Moreno J. & Alvar J. 2002. Canine leishmaniasis: epidemiological risk and the experimental model. Trends Parasitol, 18(9), 399–405. https://doi. org/10.1016/s1471‑4922(02)02347‑4 Moreno J., Nieto J., Chamizo C., González F., Blanco F., Barker D.C. & Alvar J. 1999. The immune response and PBMC subsets in canine visceral leishmaniasis before, and after chemotherapy. Vet Immunol Immunopathol, 71(3‑4), 181–195. https://doi. org/10.1016/s0165‑2427(99)00096‑3 Moshfe A., Mohebali M., Edrissian G., Zarei Z., Akhoundi B., Kazemi B., Jamshidi S. & Mahmoodi M. 2009. Canine visceral leishmaniasis: asymptomatic infected dogs as a source of L. infantum infection. Acta Trop, 112(2), 101–105. https://doi.org/10.1016/j.actatropica.2009.07.004 Mümtaz, G. 2018. An Overview of Leishmaniasis: Historic to Future Perspectives. IntechOpen. https://doi.org/10.5772/intechopen.81643 Organización Mundial de la Salud (OMS). 2019. Leishmaniasis. Marzo de 2019. https://www. w h o. i n t / e n / n e w s ‑ ro o m / f a c t ‑ s h e e t s / d e t a i l / leishmaniasis Oryan A. & Akbari M. 2016. Worldwide risk factors in leishmaniasis. Asian Pac J Trop Med, 9(10), 925‑ 932. https://doi.org/10.1016/j.apjtm.2016.06.021 Gracia M.J. & Castillo J.A. 2000. Experimental infection of Phlebotomus perniciosus and determination of the natural infection rates of Leishmania infantum in dogs. Acta Trop, 77(2), 203–207. https://doi.org/10.1016/s0001‑ 706x(00)00141‑8 Guzmán‑Barragán B.L., Ballesteros‑González C., Torres‑González D. & Guzman Y. L. 2021. Brote inusitado de leishmaniasis cutánea en zona rural de Ibagué: desafíos de la notificación. Rev UDCA Actual Divulg Cient, 24(1). http://doi.org/10.31910/ rudca.v24.n1.2021.1502 Kearse M., Moir R., Wilson A., Stones‑Havas S., Cheung M., Sturrock S., Buxton S., Cooper A., Markowitz S., Duran C., Thierer T., Ashton B., Meintjes P. & Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28(12), 1647–1649. https://doi.org/10.1093/bioinformatics/bts199 Lachaud L., Marchergui‑Hammami S., Chabbert E., Dereure J., Dedet J.P. & Bastien P. 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol, 40(1), 210‑215. https://doi.org/10.1128/ JCM.40.1.210‑215. Lombardo G., Pennisi M.G., Lupo T., Migliazzo A., Caprì A. & Solano‑Gallego L. 2012. Detection of Leishmania infantum DNA by real‑time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Vet Parasitol, 184(1), 10–17. https://doi.org/10.1016/j. vetpar.2011.08.010 Macedo T. & Urrego D 2020. Mesa intersectorial de vectores [Conference presentation]. Secretaría de Salud Municipal de Ibagué. 11/20/2020. Manna L., Vitale F., Reale S., Caracappa S., Pavone L.M., Morte R.D., Cringoli G., Staiano N. & Gravino A.E. 2004. Comparison of different tissue sampling for PCR‑based diagnosis and follow‑up of canine visceral leishmaniosis. Vet Parasitol, 125(3‑4), 251– 262. https://doi.org/10.1016/j.vetpar.2004.07.019 Marcondes M. & Day M.J. 2019. Current status and management of canine leishmaniasis in Latin America. Res Vet Sci, 123, 261‑272. https://doi. org/10.1016/j.rvsc.2019.01.022 Membrive N.A., Rodrigues G., Gualda K.P., Bernal M.V.Z., Oliveira D.M., Lonardoni M.V.C. & Silveira T.G.V. 2012. Environmental and animal characteristics as factors associated with American cutaneous leishmaniasis in rural locations with presence of dogs, Brazil. PLoS One, 7(11), e47050. https://doi.org/10.1371/journal.pone.0047050 Mendigaña F.A 2019. Plan estratégico leishmaniasis 2018‑2022. https://www.minsalud.gov.co/sites/ Leishmania spp. in dogs, Central West Colombia Leidy Alejandra Giraldo-Martínez et al. 388 Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 Reis L.E., Coura‑Vital W., Roatt B.M., Bouillet L.É., Ker H.G., Fortes R.C., Resende D., Carneiro M., Giunchetti R.C., Marques M.J., Carneiro C.M. & Reis A.B. 2013. Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection. Vet Parasitol, 197(3‑4), 498–503. https://doi. org/10.1016/j.vetpar.2013.07.006 Ribeiro R.R., Michalick M.S.M., da Silva M.E., dos Santos C.C.P., Frézard F.J.G. & da Silva S.M. 2018. Canine leishmaniasis: an overview of the current status and strategies for control. Biomed Res Int, 2018, 1‑12. https://doi.org/10.1155/2018/3296893 Salgado‑Almario J., Hernández C.A. & Ovalle C. 2019. Geographical distribution of Leishmania species in Colombia, 1985‑2017. Biomédica, 39(2), 278‑ 290. https://doi.org/10.7705/biomedica.v39i3. 4312 Santaella J., Ocampo C.B., Saravia N.G., Méndez F., Góngora R., Gomez M.A. & Quinnell R.J. 2011. Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia. Am J Trop Med Hyg, 84(5), 674‑680. https://doi.org/10.4269/ ajtmh.2011.10‑0159 Schönian G., Nasereddin A., Dinse N., Schweynoch C., Schallig H.D., Presber W. & Jaffe C.L. 2003. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis, 47(1), 349‑58. https://doi. org/10.1016/s0732‑8893(03)00093‑2. Shang L.M., Peng W.P., Jin H.T., Xu D., Zhong N., Wang W., Wu Y. & Liu Q. 2011. The prevalence of canine Leishmania infantum infection in Sichuan Province, southwestern China detected by real time PCR. Parasit Vectors, 4, 173. https://doi. org/10.1186/1756‑3305‑4‑173 Sierra D., Vélez I.D. & Uribe S. 2000. Identificación de Lutzomyia spp. (Diptera: Psychodidae) grupo verrucarum por medio de microscopía electrónica de sus huevos. Rev Biol Trop, 48, 615‑22 Silva R.B.S., Porto M.L., Barbosa W.D.O., Souza H.C.D., Marques N.F.D.S.P., Azevedo S.S., Andrade P. & Melo M.A.D. 2018. Seroprevalence and risk factors associated with canine visceral leishmaniasis in the State of Paraíba, Brazil. Rev Soc Bras Med Trop, 51(5), 683‑688. https://doi.org/10.1590/0037‑ 8682‑0429‑2017 Sistema de Vigilancia en Salud Pública (SIVIGILA) (2019). Leishmaniasis cutánea, periodo epidemiológico VI, Colombia. https://www.ins. gov.co/buscador‑eventos/Informesdeevento/ LEISHMANIASIS%20CUT%C3%81NEA%20PE%20 VI%202019.pdf Soares M.R., de Mendonça I.L., do Bonfim J.M., Paradies P., Capelli G., Cafarchia C., De Caprariis D., Sasanelli M. & Otranto D. 2006. Incidences of Canine Leishmaniasis in an Endemic Area of Southern Italy. J Vet Med B, 53(6), 295–298. https:// doi.org/10.1111/j.1439‑0450.2006.00964.x Pardo R., Ferro C., Lozano G., Lozano C., Cabrera O. & Davies C. 1999. Flebótomos (Diptera: Psychodidae) vectores de leishmaniasis cutánea y sus determinantes ecológicos en la zona cafetera del Departamento del Huila. In Memorias, XXVI Congreso de la Sociedad Colombiana de Entomología, Santa Fé de Bogotá, Colombia,147‑63. Pardo R.H., Cabrera O.L., Becerra J., Fuya P. & Ferro C. 2006. Lutzomyia longiflocosa, posible vector en un foco de leishmaniasis cutánea en la región subandina del departamento del Tolima, Colombia, y el conocimiento que tiene la población sobre este insecto. Biomédica, 26(Suppl. 1), 95‑108. Paternina‑Gómez M., Díaz‑Olmos Y., Paternina L.E. & Bejarano E.E. 2013. Alta prevalencia de infección por Leishmania (Kinetoplastidae: Trypanosomatidae) en caninos del norte de Colombia. Biomédica, 33(3). https://doi. org/10.7705/biomedica.v33i3.780 Quinnell R.J., Courtenay O., Garcez L.M., Kaye P.M., Shaw M.A., Dye C. & Day M.J. 2003. IgG subclass responses in a longitudinal study of canine visceral leishmaniasis. Vet Immunol Immunopathol, 91(3‑ 4), 161–168. https://doi.org/10.1016/s0165‑ 2427(02)00311‑2 Ramírez C.A., Requena J.M. & Puerta C.J. 2011. Identification of the HSP70‑II gene in Leishmania braziliensis HSP70 locus: genomic organization and UTRs characterization. Parasit Vectors, 4, 166. https://doi.org/10.1186/1756‑3305‑4‑166 Ramírez J.D., Hernández C., León C.M., Ayala M.S., Flórez C. & González C. 2016. Taxonomy, diversity, temporal and geographical distribution of Cutaneous Leishmaniasis in Colombia: A retrospective study. Sci Rep, 6, 28266. https://doi. org/10.1038/srep28266 Reale S., Maxia L., Vitale F., Glorioso N.S., Caracappa S. & Vesco G. 1999. Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood. J Clin Microbiol, 37(9), 2931–2935. https:// doi.org/10.1128/JCM.37.9.2931‑2935.1999 Reis A.B., Martins‑Filho O.A., Teixeira‑Carvalho A., Giunchetti R.C., Carneiro C.M., Mayrink W., Tafuri W.L. & Corrêa‑Oliveira R. 2009. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Vet Immunol Immunopathol, 128(1‑3), 87–95. https://doi. org/10.1016/j.vetimm.2008.10.307 Leidy Alejandra Giraldo-Martínez et al. Leishmania spp. in dogs, Central West Colombia Veterinaria Italiana 2022, 58 (4), 379-389. doi: 10.12834/VetIt.2632.16820.2 389 Travi B.L., Tabares C.J., Cadena H., Ferro C. & Osorio Y. 2001. Canine visceral leishmaniasis in Colombia: relationship between clinical and parasitologic status and infectivity for sand flies. Am J Trop Med Hyg, 64(3), 119‑124. https://doi.org/10.4269/ ajtmh.2001.64.119 Trevisan D.A.C., Lonardoni M.V.C. & Demarchi I.G. 2015. Diagnostic methods to cutaneous leishmaniasis detection in domestic dogs and cats. An Bras Dermatol, 90(6), 868‑872. https://doi. org/10.1590/abd1806‑4841.20153716 Verçosa B.L.A., Lemos C.M., Mendonca I.L., Silva S.M.M.S., De Carvalho S.M., Goto H. & Costa F.A.L. 2008. Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis. BMC Vet Res, 4(1), 1‑7. https://doi. org/10.1186/1746‑6148‑4‑45 Wang J.Y., Ha Y., Gao C.H., Wang Y., Yang Y.T. & Chen H.T. 2011. The prevalence of canine Leishmania infantum infection in western China detected by PCR and serological tests. Parasit Vectors, 4(1), 69. https://doi.org/10.1186/1756‑3305‑4‑69 Zoghlami Z., Chouihi E., Barhoumi W., Dachraoui K., Massoudi N., Helel K.B. & Gharbi M. 2014. Interaction between canine and human visceral leishmaniases in a holoendemic focus of Central Tunisia. Acta Trop, 139, 32‑38. https://doi. org/10.1016/j.actatropica.2014.06.012 Rodrigues J.A., Werneck G.L. & Costa C.H. 2011. Canine visceral leishmaniasis in Teresina, Brazil: Relationship between clinical features and infectivity for sand flies. Acta Trop, 117(1), 6–9. https://doi.org/10.1016/j.actatropica.2010.08.015 Solano‑Gallego L., Morell P., Arboix M., Alberola J. & Ferrer L. 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. J Clin Microbiol, 39(2), 560–563. https://doi.org/10.1128/JCM.39.2.560‑ 563.2001 Sundar S. & Rai M. 2002. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol, 9(5), 951–958. https://doi.org/10.1128/cdli.9.5. 951‑958.2002 Thrusfield M. 2018. Veterinary epidemiology (4th ed). John Wiley & Sons. ISBN: 978‑1‑118‑28028‑7 Torres E., Quintanilla M.R., Ruiz J. & Arenas R. 2017. Leishmaniasis: a review. F1000Res, 6. https://doi. org/10.12688/f1000research.11120.1 Toz S.O., Culha G., Zeyrek F.Y., Ertabaklar H., Alkan M.Z., Vardarlı A.T., Gunduz C. & Ozbel Y. 2013. A real‑time ITS1‑PCR based method in the diagnosis and species identification of Leishmania parasite from human and dog clinical samples in Turkey. PLoS Negl Trop Dis, 7(5), e2205. https://doi. org/10.1371/journal.pntd.0002205