The first case of canine visceral leishmaniasis in the southern region of Santa Catarina, an emerging focus of visceral leishmaniasis in Brazil: regional report or reflection of the reality of a country?

Visceral leishmaniasis is among the most neglected diseases in the world and especially affects poor populations of some developing or underdeveloped countries, with human disease being a factor in the occurrence of thousands of deaths every year. Visceral leishmaniasis affect domestic dogs, main reservoirs of the Leishmania parasite in urban environments. Thus, the aim of this study was to analyze the prevalence of canine visceral leishmaniasis in two non-endemic municipalities located in southern Santa Catarina, as well as to identify the main risk factors for infection in dogs. A cross-sectional study was carried out with dogs living in two Zoonoses Control Centers located in the cities of Tubarão and Criciúma. Blood samples were collected and analyzed by real-time PCR for detection of Leishmania infection. Clinical examination and evaluation of the characteristics of the canine population were also carried out. A total of 107 dogs were evaluated, of which 19% were positive for Leishmania , and 85% of the positive cases were asymptomatic. In conclusion, we highlight the detection of cases of canine visceral leishmaniasis in two cities that had not yet reported cases. The high rate of positive asymptomatic animals suggests the maintenance of the parasite in the environment.


Introduction
Visceral leishmaniasis (VL) is an infectious disease, caused by Leishmania protozoan transmitted to humans and canids through the infecting bite of female phlebotomine sandflies (Ministério da Saúde, 2009). In Brazil and worldwide, VL is considered one of the most important parasitic protozoan diseases for public health due to its high morbidity and mortality, as well as the large number of unreported cases (Gontijo & Melo, 2004). In Brazil this disease is caused by Leishmania infantum and its main vector is the phlebotomine Lutzomyia longipalpis (Michalsky et al., 2002).
Until the 1990s, about 90% of VL cases throughout Brazil were concentrated in the Northeast Region. However, in recent years the disease has begun to expand to the North, Midwest and Southeast regions of Brazil (Werneck, 2010), with emphasis on the urbanized profile of VL recently evidenced, for example, in Minas Gerais (2020) (da Silva et al., 2020). The Development, v. 10, n. 17, e167101724326, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i17.24326 3 Southern region recorded the lowest number of cases in the whole country (Martins-Melo et al., 2014), and until 2007 it was considered without risk of transmission to VL, when only a few imported human cases have been reported (Ministério da Saúde, 2010). However, there is an increase in the number of VL cases in the south region, as observed in the states of Rio Grande do Sul and Paraná, and even in some regions of Argentina. Until 2006, southern Brazil, Argentina and Uruguay were considered non-endemic areas. After outbreaks of VL in the city of Asunción, Paraguay, the first autochthon case of human VL in Argentina was reported in the city of Posadas, bordering Paraguay (Salomon et al., 2008;Acardi & Liotta, 2010). In 2009, the first outbreak of VL occurred in southern Brazil, was in São Borja, RS, a city bordering Argentina, 160 km from Posadas (Deboni et al., 2011). Also in Rio Grande do Sul, an epidemiological study by , showed positive cases of canine leishmaniasis in three cities in the metropolitan region of Porto Alegre, concluding a prevalence of 4% . Santa Catarina, in particular, was a state considered VL-free until 2012, when Figueiredo et al. (2012) reported the first autochthone cases of canine visceral leishmaniasis (CVL) registered in Florianopolis, the capital of Santa Catarina (Figueiredo et al., 2012).
Although certain measures established for the control of the disease may reduce transmission for a long period of time, there are studies indicating that about 80% of infected dogs from endemic areas can exhibit the asymptomatic form of leishmaniasis (Dantas-Torres & Brandão-Filho, 2006). Thus, these animals act as active sources of infection and go unnoticed by current serological tests, as already described by Carvalho et. al (2018), who compared serological tests and the PCR technique, detecting Leishmania DNA in asymptomatic dogs that had negative results in serology. The use of molecular techniques in the search for the prevalence of CVL in non-endemic areas of VL can provide important data to Brazilian health systems and contribute to disease prevention strategies in humans, since canine infection precedes disease in humans (Marzochi et al., 2009). Thus, the aim of this study was to analyze the prevalence of canine visceral leishmaniasis in two nonendemic municipalities located in southern Santa Catarina, as well as to identify the main risk factors for infection in dogs.

Data collection
For this study, blood of sheltered dogs from two ZCC's were evaluated for CVL. The animals were previously identified with collars containing an identification number, or by the registration number of a local non-governmental organization. Data collection regarding the evaluated animals was performed from the completion of a survey to obtain the following information: date of application, identification of the animal, municipality, race, type of pelage, age, sex, reproductive status, state and municipality of their origin, and responsible for the animal (Non-governmental organization, Caregiver, ZCC's, others). Also, current home time and location (urban/rural), animal health conditions (healthy/suspicious), being considered suspected clinical signs of leishmaniasis: dermatopathies in general, keratoconjunctivitis, progressive slimming and onicogrifosis. This questionnaire was completed through anamnesis and by clinical observation of the dogs by a veterinarian.

Samples
The samples were defined by means of a census, where all the animals' inhabitants of the sites were included.
Animals with lower volume of blood collected than that necessary for analysis (5 ml) were excluded. In the collections, all animals living in places with higher concentration of dogs from each municipality were included, being healthy or with CVL symptoms, under the responsibility of the ZCC's. A 5ml of blood and a 2.5ml sample were collected from each dog and stored in a 2.6ml S-Monovette tube containing EDTA. The other sample was placed in a 2.6 ml S-Monovette tube with clot activator and stored in a refrigerator at a temperature of 2 to 8°C for a maximum of 24 hours. The contents of the tubes were centrifuged in centrifuge Fanem Excelsa Baby II at 3500 rpm for 10 minutes to obtain the serum or plasma, according to the material Research, Society and Development, v. 10, n. 17, e167101724326, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i17.24326 5 collected. The material resulting from centrifugation was stored in Eppendorf tubes and packed at -20º C.

Real-time PCR amplification
For the identification of dogs positive for Leishmania infection, the DNA was isolated from dog serum samples (200   μl For each amplification, a negative control (reaction mix) was used and purified Leishmania DNA was used as a positive control. All samples were made in duplicate as a form of evidence. The sample was defined as positive when it had a detectable cycle threshold (Ct) and the melting temperature (Tm) was the same as for the positive control (Rolim et al., 2016).
The amplification and dissociation curves were analysed using the StepOne™equipment software.

Data processing and analysis
Pearson's chi-square method, Fisher's exact test and likelihood ratio were used for statistical analysis, according data available. All analyses were performed using the software OpenEpi 3.01 and SPSS 21.0.

Ethical principles
This study was approved by the Ethics Committee on the Use of Animals -CEUA/UNISUL to assess compliance with Normative Resolution No. 12 of September 20, 2013, under no. 16.050.2.13.IV.

Results
A total of 107 dog samples were analyzed, of which 77 dogs were from Tubarão (72%) and 30 from Criciúma (28%).

Discussion
This study evaluated the prevalence of CVL in shelter dogs from two municipalities in southern Santa Catarina, using the qPCR technique to detecting the Leishmania DNA. The results showed that the prevalence of infection in the total number of animals evaluated (n=107) was 19% between the two municipalities studied, 16.9% in Tubarão and 23.3% in Criciúma. These regions had not yet registered any cases of the disease in dogs, although some cases have already been registered in other regions of Santa Catarina. In Florianopolis Steindel et al., (2013), evaluated dogs with suspected Leishmania infection between 2010 and 2011 and found positive animals with a prevalence of 1.4%. In the western region of this state, As in Santa Catarina, in areas previously considered non-endemic for CVL in other states of Brazil (Steindel et al., 2013;Maziero et al., 2014), showed a similar situation when using the PCR technique for the testing of dogs as recorded in some municipalities of Rio de Janeiro (De Paula et al., 2009;Campos et al., 2013) and Minas Gerais (Castro et al., 2019). In the metropolitan region of Porto Alegre (RS), a non-endemic region, a prevalence of 4% was observed through real time PCR and serology, being the essential molecular method for excluding false negatives and false positives . It is very likely that CVL transmission is already occurring among dogs in other municipalities located in the south of Santa Catarina, increasing the chances of human infection (Ministério da Saúde, 2009). In relation to the tests to evaluate CVL infection, there are many variations in the sensitivity and specificity of serological methods. So, it is important to perform confirmatory tests to establish the diagnosis of CVL in non-endemic areas. However, it is important to note that even using tests with lower specificity, cases of CVL in non-endemic regions were verified, such as in Florianopolis-SC (Corrêa et al., 2010), Joinville-SC (Pacheco et al., 2013) and Poxoreo-MT (Azevedo et al., 2008).
In relation to the phlebotomine, in 2017, a survey of sandflies fauna was carried out in southern Santa Catarina, and its presence was identified (Variza et al., 2021), corroborating the high prevalence of CVL described in our results. The association between the occurrence of the parasite and the presence of the vector in the same environment favors conditions for the establishment of the disease. Therefore, it is a matter of time for the appearance of human cases, if there are no vector and canine reservoir control.
According to the Brazilian Ministry of Health (2006), dogs cohabiting urban areas have a higher rate of positivity for Leishmania than those from rural areas in several regions of Brazil. In Santa Catarina, this pattern seems to be confirmed.
With the increased incidence of CVL in urban areas, associated with the sinantropism of some species of sandflies and the spread of reservoir hosts to anthropic environments (González et al., 2014), the State of Santa Catarina needs urgent care by public health agencies. In a negative way the population is subject to become infected and the region endemic in CVL.
Regarding the risk factors associated with dogs, there was a higher prevalence among animals older than seven years (33.3%), followed by animals up to 11 months old (20.6%). Similar results were also reported by França- Silva et al., (2003).
However, advanced age does not seem to be a risk factor, since other studies suggest that younger animals pose a higher risk for CVL (Moreira et al., 2003). As in others studies (França-Silva et al., 2003;Silva et al., 2018). we also found no significant association between gender and prevalence of CVL.
From the positive animals, 85% were asymptomatic, which may have been a great reason for the dissemination and maintenance of CVL in the region. It is known that the dogs, even without symptoms, can have high parasitic load and act as a reservoir of the parasite, having an important role in the transmission through the vector (Steindel et al., 2013). Also, as reported in other study (Azevedo et al., 2008), skin lesions were the most frequently observed clinical signs in seropositive and symptomatic animals. The study presents limitations inherent to a cross-sectional approach, such as the impossibility of verifying causality. It should be considered the limitation that the form of study was carried out by census of zoonosis centers, not knowing for sure which specific location this dog belonged to, nor if the contamination occurred in the city or in the kennel itself.

Conclusion
In conclusion, we highlight the detection of cases of CVL in two cities that had not yet reported cases. It was not possible to define any significance among the proposed variables as risk factors for infection. However, the high rate of positive asymptomatic animals suggests the maintenance of the parasite in the environment. There are numerous socioeconomic and scientific challenges that could prevent the success of the control and prevention of this disease in all regions. Efforts and research to understand the dynamics of CVL transmission in the region are necessary to avoid the expansion of canine cases and the consequent human infection.