Survey of protozoan vector-borne diseases in dogs from Atlantic Rainforest fragment around Billings Dam, São Paulo, Brazil

ABSTRACT: Vector-borne diseases are currently one of the biggest public health concerns worldwide. Dogs, being the closest companion animals to humans, are considered the main reservoir of some of these diseases in the urban environment. Therefore, the study of the disease behavior in dogs can help to understand the disease affecting human health. Serological and molecular diagnoses of Babesia vogeli, Rangelia vitalli, Leishmania infantum, and other trypanosomatids, were performed by immunochromatographic and PCR assays, respectively, on dogs in a dog shelter located in an Atlantic Forest fragment near the Billings Dam, São Bernardo do Campo, São Paulo-Brazil. Our molecular diagnostic results showed a high prevalence of Babesia vogeli, at 20.9% (17/81). No other protozoan was detected in any of the tests. Determining the prevalence of major vector-borne diseases is essential to establish preventive and control measures for zoonotic diseases in animals kept in shelters, in order to minimize the impact of vector-borne diseases on animal health.


INTRODUCTION
Vector-borne diseases are one of the biggest public health problems (DANTAS-TORRES, 2008;MAGGI et al., 2019). Dogs, being the closest companion animals to humans, are considered the main reservoir of some of these diseases in the urban environment, and studying the behavior of the disease in dogs can help in our understanding of the disease as a whole and of how it integrates within human health (DANTAS-TORRES, 2008;DANTAS-TORRES et al., 2014).

Visceral leishmaniosis, caused by
Leishmania infantum, is now considered one of the most serious vector-borne diseases. In Brazil, it is responsible for 90% of all human and canine (PAHO, 2019). Lutzomyia longipalpis is the main vector, which is widely distributed (KRAUSPENHAR et al., 2003;MONGRUEL et. al., 2018), but other species of this genus are also considered vectors (WALKER et al., 2000;MORAES-FILHO et al., 2011;DANTAS-TORRES et al., 2019).
Canine babesiosis is a hemolytic disease that, in Brazil, is caused by Babesia vogeli, in Azevedo et al. which all clinical signs of the disease are caused by erythrocyte lysis (SOARES et al., 2018;DANTAS-TORRES et al., 2019). It is transmitted by the tick Rhipicephalus sanguineus sensu lato, which has a cosmopolitan distribution, nesting behavior, and easily adapts to changes in temperature and humidity (WALKER et al., 2000;MORAES-FILHO et al., 2011;DANTAS-TORRES et al., 2019).
Rangelia vitalli, the causative agent of canine rangeliosis, is a protozoan piroplasma that infects erythrocytes, leukocytes, and endothelial cells in dogs (CARINE et al., 1914;FIGHERA et al., 2010;MONGRUEL, 2018). It is more common in wild canids because its unique vector, the tick Amblyomma aureolatum, is exclusively found in areas of the Atlantic rainforest where the temperature and humidity conditions are favorable for its development (SOARES et al., 2018;DANTAS-TORRES et al., 2019).
In this study, we performed serological and molecular diagnoses of some protozoans with importance to health that are transmitted by different arthropods, such as Babesia vogeli, Rangelia vitalli, and Leishmania infantum, in a dog shelter located in an Atlantic Forest fragment in the influential area of Billings Dam in the Riacho Grande district, São Bernardo do Campo, São Paulo-Brazil.

Geographical area, study population, and sample collection
This study was conducted in a private shelter located in the Riacho Grande district of the city of São Bernardo do Campo in the metropolitan region of São Paulo, located at 23°48′ 11.2″ S and 46°32′ 07.6″ W (Figure 1). The study area is an Atlantic Forest fragmentation close to the Billings Dam. The kennels are rustic, made of wood, and the area for dogs is surrounded by the native Atlantic Rainforest of the region. Dogs in the shelter were mixed-breed animals that had been abandoned nearby, dogs that were born in the shelter from pregnant bitches, dogs that had been adopted more than once and returned due to lack of adaptation to the adopted home, and even purebred species that were part of clandestine kennels or were abandoned because they were born with some deformity or disability. It was not possible to obtain parameters to measure the age of the dogs. Peripheral whole blood was collected from all 81 dogs that lived in the shelter on January 15, 2019. All dogs were inspected for the presence of ectoparasites and samples were collected.
In dogs that tested positive for serological and molecular markers of disease, fine needle aspiration of the popliteal lymph nodes was performed to isolate the parasite.

Serological screening
Serological screening was performed on peripheral whole blood immediately after collection by immunochromatographic assay, using the DPP © Rapid Diagnostic Kit for Canine Leishmaniosis (Biomanguinhos/Fiocruz) for the detection of K9/K26/K39-reactive antibodies, according to manufacturer's instructions.

DNA extraction
For DNA extraction, the commercial PureLink Genomic DNA Mini Kit (Invitrogen) was used according to the manufacturer's instructions.

Molecular markers for diagnosis
For the specific molecular diagnosis of Leishmania infantum, PCR was performed using L-like Cathepsin genes  and ribosomal internal transcribed spacer (ITS) (SCHONIAN et al., 2003) as targets.
For detection of other trypanosomatid species, the V7V8 region of SSU rDNA was used, as this is considered the trypanosomatid barcode (MAIA DA SILVA et al., 2004). The characterization of isolates was performed by sequencing SSU rDNA and gGAPDH (MARCILI et al., 2014).

Isolation and culture of Leishmania parasites
For dogs positive by serological and/ or molecular examination, parasite isolation was performed by fine needle aspiration of popliteal lymph nodes (MARCILI et al., 2014). Samples were inoculated into tubes of biphasic medium consisting of BAB (blood agar base, 15% rabbit blood) as the solid phase and LIT or RPMI medium, containing fetal bovine serum and antibiotics, as the liquid phase.

RESULTS
The 81 samples were submitted for Leishmania infantum serology by the DPP © Rapid Test distributed by the Brazilian Ministry of Health, and 2.46% (2/81) were positive.
Molecular tests for the trypanosomatid barcode region (V7V8 SSU rDNA) and the gGAPDH gene were negative for all samples. Specific molecular markers for the diagnosis of the Leishmania genus (ITS1 SSU rDNA) and L. infantum species (cathepsin L-like) were also negative for all samples tested.
Additionally, fine needle aspiration of the popliteal lymph nodes in dogs that had a positive reaction to the DPP© test was performed to attempt to isolate parasites. All cultures were negative for trypanosomatid parasites. The molecular diagnosis of Rangelia vitalli was also negative for all samples tested. A total of 20.9% (17/81) of the samples were positive for Babesia vogeli. No ectoparasites were found in the examined dogs.

DISCUSSION
In the present study, 20.9% of dogs were positive for Babesia vogeli, which is much higher than previous studies conducted in São Paulo and other locations in the Southeast region of Brazil (SILVA et al., 2014;O'DWYER et al., 2009;MONGRUEL et al., 2018). It is known that high canine population density favors both an increase in the number of R. sanguineus ticks and their permanence in the environment, as well as pathogen circulation in the population. A study conducted in a dog shelter on Rickettsia rickettsii (PACHECO et al., 2011) demonstrated an increase in both the vectors and agent in the studied population, corroborating the results obtained in this study. However, during sample collection the dogs did not have a tick infestation.
Vector-borne diseases are important in human and animal health worldwide, with cycles involving vertebrates, invertebrates, and their interaction with the environment concerning different levels of anthropic action (ALBERIGI et al., 2019). The São Bernardo do Campo municipality is a predominant region of the Atlantic Forest fragments (SÃO BERNARDO DO CAMPO, 2019).
The Atlantic Rainforest has climatic conditions favorable for the development of Amblyomma spp. and phlebotomine (PINTER et al., 2004;SCINACHI et al., 2017); however, positive results were observed only for parasites of the genus Babesia, which is transmitted by ticks of the species R. sanguineus that are nidicolous and closely associated with canines in urban areas (GALAY et al., 2018).
In forested areas, adult A. aureolatum ticks feed on wild carnivores (GUGLIEMONE et al., 2003), Azevedo et al. whereas in rural areas near forests, these ticks feed on domestic canines, enabling the transfer of these ectoparasites from dogs to humans (GUGLIEMONE et al., 2003;PINTER et al., 2004). A. aureolatum is the only species capable of transmitting R. vitalli, thus all clinical cases are related to an infestation of this tick (SOARES et al., 2018;. All dogs sampled in this study were kept in enclosures without access to the woods around the shelter, which corroborates the negative results for R. vitalli. Positive results of serological tests for Leishmania may result from cross-reactions with other pathogens, most commonly with other trypanosomes, such as Trypanosoma caninum (ALVES et al., 2012), and leishmania vaccines (SOLANO-GALEGO et al., 2017;MARCONDES et al., 2013). This may account for the positive serological tests in this study, as the parasitological diagnosis was negative for serologically positive dogs. In addition, the molecular diagnosis was negative for all samples based on cathepsin L-like and ITS SSU rDNA markers. Despite the low parasitemia presented in Leishmania infantum infections, the use of the whole blood instead of the poplitel lymph node samples for molecular diagnosis is based to the high sensitivity and specificity of the marker based on Catepsin L-like.
The canine population in the city of São Paulo was estimated at 2,507,401 in 2012, with a human:dog ratio of 4.34, and a higher concentration of dogs in urban areas than rural areas of the municipality (CANATTO et al., 2012). The proportion of dogs adopted (73.9%) was significantly higher than that of dogs purchased (26.1%), and the frequency of dog adoption is correlated with the frequency of households with a dog and greater levels of social exclusion (CANATTO et al., 2012). In this context, dog shelters play an important role in dog care and the adoption process.
Sanitation guidelines for animals kept in shelters should be proposed to minimize the impact of vector-borne diseases on animal health, as well as for the prevention and control of zoonotic diseases.

CONCLUSION
This study showed that Babesia vogeli is highly prevalent in dogs from the Atlantic Rainforest fragment around Billings Dam, São Paulo-Brazil. These results also emphasized the need to test for multiple vector-borne diseases in dogs suspected of infection, to facilitate appropriate treatment and to promote the adoption of animals abandoned and kept in shelters.