Vector-borne pathogens of zoonotic concern in dogs from a Quilombola community in northeastern Brazil

Canine vector-borne pathogens (CVBPs) comprise a group of disease agents mainly transmitted by ticks, fleas, mosquitoes and sand flies. In this study, we assessed the presence of CVBPs in an Afro-descendent community (Quilombola) of northeastern, Brazil. Dog blood samples (n = 201) were collected and analyzed by rapid test for the detection of antibodies against Leishmania spp., Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi sensu lato (s.l.), and antigens of Dirofilaria immitis. In addition, polymerase chain reactions were performed for Anaplasmataceae, Babesia spp., Hepatozoon spp., Rickettsia spp. and B. burgdorferi s.l. Overall, 66.7% of the dogs scored positive to at least one pathogen at serological and/or molecular methods. Antibodies against Ehrlichia spp. were the most frequently detected (57.2%; n = 115/201), followed by Anaplasma spp. (8.5%; n = 17/201), Leishmania spp. (8.5%; n = 17/201) and B. burgdorferi s.l. (0.5%; n = 1/201). For D. immitis, 11 out of 201 (5.5%) animals scored positive. At the molecular analysis, 10.4% (n = 21/201) of the samples scored positive for Babesia spp./Hepatozoon spp., followed by Anaplasmataceae (5.0%; n = 10/201) and Rickettsia spp. (3.0%; n = 6/201). All samples were negative for B. burgdorferi s.l. Our data demonstrated the presence of CVBPs in the studied population, with a high seropositivity for Ehrlichia spp. In addition, considering the detection of zoonotic pathogens in dogs and their relationship with people from Quilombola communities, effective control strategies are advocated for minimizing the risk of infection in this socially vulnerable human population and their pets.


Introduction
Canine vector-borne diseases (CVBDs) are caused by a group of pathogens mainly transmitted by ticks (e.g., Babesia spp., Ehrlichia spp., Anaplasma spp., Rickettsia spp.), fleas (e.g., Rickettsia felis, Dipylidium caninum), mosquitoes (e.g., Dirofilaria spp.) and sand flies (e.g., Leishmania spp.) (Otranto 2018;Maggi and Krämer 2019;Alhassan et al. 2021). The zoonotic potential of some CVBD-causing agents represents a threat to human health, especially in the tropics and subtropics, as the climatic conditions of these areas are conducive to the growth and proliferation of arthropod vectors (Dantas-Torres 2008; Maggi and Krämer 2019). The CVBDs of major veterinary and public health concern are leishmaniasis and heartworm disease (HWD), followed by anaplasmosis, ehrlichiosis and babesiosis Borges et al. 2021). In Brazil, dogs can be infected by zoonotic Leishmania spp., including Leishmania infantum, Leishmania amazonensis and Leishmania braziliensis (Belo et al. 2013;Souza et al. 2019). Leishmania spp. infection occurs mostly through the bites of infected female phlebotomine sand flies (Alvar et al. 2012). Leishmaniases are still predominantly rural diseases, with wildlife species (e.g., foxes, opossums and rodents) acting as principal hosts of the parasites (Roque and Jansen 2014;Bezerra-Santos et al. 2021). However, dogs are considered the main reservoirs of L. infantum and source of infection to the vectors in both rural and urban areas (Dantas-Torres 2007). Another important parasite of zoonotic potential in Brazil is Dirofilaria immitis, the agent of HWD, often characterized by severe cardiopulmonary disorders which may be fatal. The distribution of D. immitis overlaps that of mosquito vectors of the genera Culex, Aedes, Ochlerotatus and Anopheles (Ahid and Lourenço-Oliveira 1999;Simón et al. 2012;Ramos et al. 2016;Maggi and Krämer 2019;Brianti et al. 2021;Mendoza-Roldan et al., 2021). For example, a previous study demonstrated the presence of competent vectors of D. immitis (e.g., Ochlerotatus scapularis and Ochlerotatus taeniorhynchus) occurring throughout the year in an endemic area of Brazil (Labarthe et al. 1998), with a correlation between the high populations of arthropod vectors and the odds of infection in dogs and humans (Bendas et al. 2019).
Anaplasma platys, Ehrlichia canis and Babesia vogeli are the major causative agents of canine anaplasmosis, ehrlichiosis and babesiosis in Brazil (Dantas-Torres 2008), which are characterized by a variety of clinical signs (e.g., pyrexia, lethargy, weight loss, lymphadenopathy, bleeding tendencies, hemoglobinuria, petechiae, jaundice and even death) (Ybañez et al. 2018) and hematological alterations (e.g., anemia and thrombocytopenia) in infected animals. The main biological vector of these hemoparasites is the brown dog tick (i.e., the recently proposed Rhipicephalus linneai, also referred to as the tropical lineage of Rhipicephalus sanguineus sensu lato by Šlapeta et al. 2021), which has also been associated with the transmission of other pathogens, such as Hepatozoon canis and bacterial species belonging to the spotted fever group rickettsiae (SFGR) (Ramos et al. 2014;Santos et al. 2018;Alhassan et al. 2021). Animals coinfected by multiple CVBD-causing pathogens are at higher risk of developing severe clinical signs (de Caprariis et al. 2011). For example, a study estimated that dogs with leishmaniasis are 12 times more likely to be coinfected with E. canis compared to healthy ones, with increased severity of clinical presentation, and potential reduction in the efficacy of the treatment (Attipa et al. 2018).
In Brazil, there are an estimated number of about 3,000 Quilombola communities composed by descendants of slaves of African origin, usually located in remote rural areas (Conde et al. 2020). People from these communities commonly live from agriculture and/or use of forest resources (Conde et al. 2020). In these areas wandering dogs, as well as owned restricted and semi-restricted ones are usually part of the domestic fauna. Data on canine zoonotic pathogens from these communities are scarce, with a single report recording a prevalence of 8.4% for L. infantum in skin samples from dogs (Silva et al. 2017). Nevertheless, the presence of wandering dogs, vectors and deficient sanitary conditions suggest that other pathogens of veterinary and public health concern may occur in these areas. Thus, the present study was aimed to assess the occurrence of CVBD-causing pathogens in a Quilombola community in the northeastern region of Brazil.

Study area
This study was conducted in a Quilombola community in the municipality of Garanhuns (08°53′25" S, 36°29′34" W; 896 m above sea level), Pernambuco, Northeastern Brazil. Semi-restricted or unrestricted dogs are frequently present in the study area and live in contact with other domestic animals (e.g., cats, birds and domestic ruminants) and wildlife (e.g., opossums, rodents and birds). The study area has a tropical savanna climate with dry-summer characteristics, which corresponds to the Köppen climate classification category "Aw," presenting a mean annual temperature of 20 ºC (range, 16-30 ºC), relative humidity of 76% (38-100%) and precipitation of 873 mm (751-1000 mm) (Barbosa et al. 2016), with rains concentrated from April to July (Andrade et al. 2008).

Sample collection and laboratory analysis
From July 2021 to January 2022, blood samples (1.5-5 ml) were collected from cephalic vein of 201 dogs. Samples were divided into two aliquots: one stored in tubes containing EDTA, and the other in tubes without anticoagulant, for molecular and serological analysis, respectively. Data on sex, dog categories (i.e., restricted and semi-restricted according to Otranto et al. 2017), overnight stay and the presence of ectoparasites were recorded for each dog. The serum samples were tested by an immunochromatographic assay (DPP® CVL, BioManguinhos) for anti-Leishmania spp. antibodies and by an ELISA rapid test (SNAP® 4Dx Plus, Idexx), which detects antibodies to Anaplasma spp. (A. platys/A. phagocytophilum), Ehrlichia spp. (E. canis/E. ewingii), B. burgdorferi s.l. and antigens of D. immitis. All tests were performed according to the manufacturer's instructions.
Genomic DNA was extracted from 200 μl aliquots of EDTA-treated blood samples using GenUP DNA Kit (Biotechrabbit, Berlin, Germany) following the manufacturer's instructions. All samples were tested for the presence of Anaplasmataceae, Borrelia spp., Rickettsia spp., Babesia spp. and Hepatozoon spp. by conventional PCR, using the primers reported in Table 1. PCR products were purified (Thermo Scientific™ FastAP™ Thermosensitive Alkaline Phosphatase) and sequenced using the same forward and reverse primer sets reported in Table 1 for each pathogen, employing the Big DyeTerminator v.3.1 chemistry in a 3130 Genetic analyzer (Applied Biosystems, California, USA) in an automated sequencer (ABI-PRISM 377). Nucleotide sequences were edited, aligned and analyzed using Mega 7.0 software and compared with reference sequences available on GenBank database using Basic Local Alignment Search Tool (BLAST; http:// blast. ncbi. nlm. nih. gov/ Blast. cgi).

Data analysis
Descriptive statistics were used to calculate relative and absolute frequencies. Exact binominal 95% confidence intervals (CIs) were established for proportions. The chi-square with Yates correction was used to compare proportions with a p value < 0.05 regarded as statistically significant. Analyses were performed using Epitools-Epidemiological Calculators (https:// epito ols. ausvet. com. au/).
According to BLAST analysis, two samples positive for the 18S rRNA gene revealed 100% nucleotide identity with B. vogeli (accession number: MN700646), and 19 samples

Discussion
Data presented herein indicated a high overall level of exposure to vector-borne pathogens in dogs from the Quilombola community in northeastern Brazil. In particular, over 50% of the dogs were seropositive to Ehrlichia spp., which is in line with previous studies conducted in the same region (Figueredo et al. 2017;Dantas-Torres et al. 2020). This may be partly explained by the high prevalence of the brown dog tick on dogs in the study area (Santos et al. 2017;2018). The seropositivity to other pathogens (i.e., Anaplasma spp., Leishmania spp. and D. immitis) was lower as compared with a recent study conducted in an urban area of Pernambuco (Dantas-Torres et al. 2020). Differences may be related to many eco-epidemiological factors, including the level of exposure to the vectors and climatic factors. For example, in the same area a low occurrence and diversity in sand fly species throughout a one-year sampling period has been demonstrated, with Lutzomyia evandroi being the only species identified (Ubirajara Filho et al. 2020).
Among the vector-borne protozoa, H. canis was the most frequently detected molecularly (i.e., n = 19 dogs were infected by H. canis, while only two dogs by B. vogeli). This may be due to the shorter time of circulation of B. vogeli in the blood of infected animals. Epidemiological data from previous studies demonstrated that B. vogeli is highly prevalent in Brazil, with up to 8.2% molecularly positive dogs in Pernambuco (Ramos et al. 2010;Dantas-Torres et al. 2021), 8.0% in São Paulo (O'Dwyer et al. 2009), 10.0% in Paraíba (Rotondano et al. 2015), 15.0% in Ceará (Fonsêca et al. 2022), 30.6% in Minas Gerais (Barbosa et al. 2020)  and 14.1% in Rio de Janeiro (Paulino et al. 2018;Camilo et al. 2021). Conversely, H. canis has been reported with lower prevalence (i.e., 0.4% to 5.4%) in Northeastern Brazil (Ramos et al. 2010;Dantas-Torres et al. 2021), differently from the Southeastern region, in which higher prevalence values (i.e., 58.7-79.2%) were reported (Miranda et al. 2014;Spolidorio et al. 2009). The simultaneous detection of Ehrlichia spp. and Anaplasma spp. (6.5%) at PCR and serological tests agrees with previous reports in Pernambuco state in which both pathogens were the most frequently diagnosed molecularly (Ramos et al. 2010). The above picture is probably due to the high abundance of brown dog ticks, which may transmit multiple pathogens simultaneously (Shaw et al. 2001). Similarly, the positivity to both Ehrlichia spp. and Leishmania spp. is of importance due to the implications those pathogens may have in the pathogenesis of CVBD, mainly their clinical presentation, and response to therapy (de Caprariis et al. 2011;Attipa et al. 2018). For example, a study on the experimental infection of E. canis and A. platys in dogs demonstrated that these pathogens cause various changes in pathophysiological parameters (i.e., more pronounced anemia and thrombocytopenia). Therefore, raising awareness of the risks of simultaneous positivity when animals are exposed to multiple tick-borne pathogens (Gaunt et al. 2010).
Rickettsia felis, a zoonotic rickettsia belonging to the SFG, previously described infecting human patients from Kenya (Richards et al. 2010) and Serbia (Banović et al. 2021), was molecularly detected for the first time in the dogs herein assessed. This pathogen was also detected in fleas (Ctenocephalides felis) and ticks (Dermacentor nitens) collected from dogs and horses from the same study area (Oliveira et al. 2020). Rickettsia felis is widespread in Brazil, and its distribution overlaps that of C. felis, its main biological vector (Horta et al. 2014). This finding is particularly important from a One Health perspective, considering that the local circulation of zoonotic pathogens may represent an eminent risk for the Quilombola community. Funding Open access funding provided by Università degli Studi di Bari Aldo Moro within the CRUI-CARE Agreement. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability
The authors declare that data supporting the findings of this study are available within the article.

Declarations
Ethics approval The Ethics Committee for Animal Experimentation (ECAE) of Federal Rural University of Pernambuco approved all study procedures (approval number: 1041060520).
Consent to participate All dog owners read and signed an informed consent for participation in the study.

Conflicts of interest Authors declare no conflict of interest.
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