Evidence of exposure to Leptospira spp. in dogs at a zoonosis control center in Brazil

Evidence of exposure to Leptospira spp. in dogs housed in the kennel of the Zoonosis Control Center of Belém, Pará, Brazil, was investigated. Whole blood and serum samples from 145 dogs were investigated using the polymerase chain reaction (PCR) and microscopic agglutination test (MAT), respectively. A total of 64.14% of the dogs were found to be seropositive for Leptospira spp., with the most frequent serogroup being Djasiman (39.73%). However, PCR results revealed that all of the dogs were negative for Leptospira spp. DNA. Although the results of the study suggest the animals did not currently have leptospires in blood, they only show REVISTA ACADÊMICA CIÊNCIA ANIMAL 1 Laboratory of Zoonoses and Public Health, Institute of Veterinary Medicine, Universidade Federal do Pará (UFPA), Castanhal, PA, Brazil 2 Centro de Controle de Zoonoses de Belém (CCZ), Belém, PA, Brazil 3 Epidemiology and Geoprocessing Laboratory, Postgraduate Program in Animal Health in the Amazon, Institute of Veterinary Medicine, Universidade Federal do Pará (UFPA), Castanhal, PA, Brazil * Corresponding author: mirianralbuquerque@gmail.com Rev. Acad. Ciênc. Anim. 2020;18:e18301 DOI: 10.7213/2596-2868.2020.18301 ISSN: 2596-2868 Received: Jun 06 2020 | Approved: Aug 04 2020 circulating anti-Leptospira spp. antibodies, implying prior contact with the bacteria.


Introduction
Currently, zoonosis control centers aim to control the population of stray or abandoned dogs by collecting, recovering, and surgically sterilizing the dogs before making them available for responsible adoption (Miotto et al., 2018;Arruda et al., 2019).
Most of these institutions act as transit locations, with the dogs being sheltered temporarily and then adopted within a short timeframe. However, not all dogs have the privilege of being adopted, and the number of animals housed in these locations for a long time or until the end of their life is significant (Arruda et al., 2019).
These places are often lack of financial, technical, and human resources For this reason, effective health strategies are not regularly implemented (Miotto et al., 2018). This contributes to an increased risk of exposure of dogs to Leptospira spp. (Paz et al., 2015;Miotto et al., 2018).
Some studies have already shown evidence of exposure to Leptospira spp. in dogs from zoonosis control centers. Among them are the serological surveys of Oliveira et al. (2012), Paz et al. (2015) and Silva et al. (2017), which reported seroprevalences ranging from 17.41 to 53.8%, and the molecular genetic studies by Meira et al. (2011) and Oliveira et al. (2012) reporting 4% and 7.7% positive results for Leptospira spp. in blood samples, respectively.
Dogs can act as sentinels, i.e., they can signal the presence of Leptospira spp. in a given environment through evidence of exposure to the bacteria (Castro et al., 2011). Thus, they can even serve as a warning for people who may also be vulnerable to the risk of exposure (Azócar-Aedo et al., 2018).
In this context, further investigations are of great importance to improve the health of these dogs and curtail the spread of Leptospira spp. Thus, in this study, we investigated the evidence of exposure to Leptospira spp. in dogs housed in the kennel of the Zoonosis Control Center of Belém (ZCC), Pará, Brazil.

Material and methods
To calculate the sample size, the mean number of adult dogs collected by the ZCC in 2016 and 2017 (n = 230) was considered as the target population. The Statcalc tool of the EpiInfoTM7 software (CDC -Atlanta, Georgia, USA) was used. We took into consideration an expected frequency of 50%, acceptable error of 5%, and confidence interval of 95% to obtain a sample size of 144 animals. However, to prevent sample loss, 145 animals were analyzed. Convenience sampling was used in the collection of blood samples.
This study was conducted in accordance with ethical terms and conditions, and was approved by the Ethics Committee for the Use of Animals of the Federal University of Pará, Belém, Brazil (approval number: 4553100718, and publishing date: February 07, 2019).
The study was conducted at a kennel of the ZCC, Belém, Pará state, Brazil (118'58.2''S and 48º27'17.7''W). This is a government institution that among its other functions collects stray or abandoned animals that can potentially transmit zoonoses, cause traffic accidents, and attack people in different neighborhoods and districts of the municipality. Animals that are collected are dewormed, vaccinated against rabies, castrated, and if they are in good health conditions, are made available for responsible adoption by the community. All the dogs in this study were adults of unknown age, and any history of vaccination against Leptospira spp. was unknown.
From August to December 2018, blood samples were obtained by puncturing the cephalic vein with a 25.0 × 0.7 mm needle and a 5 mL syringe. The samples were stored in tubes containing gel/clot activator and tubes containing K3-EDTA to obtain serum and whole blood, respectively.
DNA from whole blood samples was extracted using the ReliaPrepTM Blood gDNA Miniprep System kit (Promega, Madison WI, USA), by following the manufacturer's recommendations.
The PCR reaction was processed in a thermal cycler (Veriti 96 Well Thermal Cycler, Applied Biosystems, Foster CA, USA) using the following conditions: an initial denaturation cycle at 94 °C for 5 min, followed by 40 amplification cycles that included denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, with the reaction being completed by an extension at 72 °C for 5 min. The positive control was DNA (5 µL) extracted from Leptospira cultivated in liquid EMJH medium serovar Can and the negative control was sterile water (5 µL). The PCR products were subjected to agarose gel electrophoresis (1.5%) and stained with GelRed® (Biotium Inc., Fremont CA, USA) for visualization under ultraviolet light of a transilluminator with a photo-documentation system (Gel DOCTM XR+ Imaging System, BioRad, Hercules CA, USA).

Results and discussion
Of the 145 serum samples examined by MAT, 64.14% (93/145) were reactive with one or more Leptospira spp. serogroups, with titers ranging from 100 to 6400 (Table 1). Twenty co-agglutinations were observed in the analyses, i.e., equal titrations for two or more serovars that made it impossible to identify the predominant serological variant. For this reason, they were not included in the frequency of serogroups. The 145 whole blood samples tested by the PCR technique for Leptospira spp. were negative.
Lower seroprevalences than the current results were reported by Oliveira et al. (2012), Paz et al. (2015) and Silva et al. (2017), who found seroprevalences ranging from 17.41% to 53.8% in dogs from ZCC in Brazil.
In the current study, the observed prevalence of dogs seropositive for Leptospira spp. may be due to the fact that before their stay in the kennel of the ZCC, these dogs lived for long periods on the streets. This exposed the dogs to a situation of vulnerability characterized by ingestion of food waste from garbage and water contaminated with bacteria, in addition to having contact with other animals such as infected synanthropic rodents (Hafemann et al., 2018).
The seroprevalence values may also have resulted from the dogs being housed in the kennel of the ZCC, where exposure to Leptospira spp. may have been favored by the difficulty of maintaining sanitary conditions due to the high density of animals and lack of financial, technical, and human resources (Miotto et al., 2018).
housed at the ZCC with infected dogs, which plays an important role in the maintenance of this serogroup. This is frequently identified as the cause of clinical and asymptomatic cases (Paz et al., 2015).
To our knowledge, this is the first study to report serological responses to the Cyn serogroup in dogs from a zoonosis control center. These findings may be associated with the possibility of contact with maintenance hosts of this serogroup. However, little is known about the species which play this role and this needs to be clarified.
The occurrence of the Ict serogroup in the analyzed animals shows that they could be exposed to infected synanthropic rodents, which are considered to be the most important reservoirs of this serogroup (Paz et al., 2015).
Reactivity to the Ser serogroup is a common finding. Its occurrence in the analyzed dogs may be related to their exposure to the street environment or even the kennels of the ZCC, where the leptospires of this serogroup are generally present, as they are saprophytes and wild (Moraes et al., 2010).
However, to ensure a reliable interpretation of serological titers, it is recommended to not rely on the analysis of a single sample, but to perform paired serological analysis with an interval of 1-2 weeks between sampling, to show an increase of four-fold or more between the obtained titers (Goldstein, 2010). Unfortunately, this procedure was not possible due to the difficulty of collecting a second blood sample from the same dog because some animals were no longer housed at the study site.
Serological responses to the Dja serogroup were predominant in the dogs analyzed; however, these responses may have been related to contact with maintenance hosts of the Dja serogroup. These results need to be further examined because of the paucity of data available on the species that effectively play this role.
Serological evidence of the Can serogroup in the analyzed dogs mainly reflects contact of dogs zoonosis control centers in Brazil. The negative molecular analysis results in our study suggest the absence of the bacterial agent (leptospiremia) in blood at the time of sampling, i.e., the dogs were probably not actively infected (Hua et al., 2016).

Conclusion
We conclude that the dogs housed in the kennel of the Zoonosis Control Center of Belém, Pará, had no leptospires in the bloodstream at the time of sampling; they were previously exposed to the bacterial agent. It is extremely important to carry out further studies in this regard, as they are still incipient in canine populations housed in shelters in this region.