HIGH LEVEL OF SEROVAR COPENHAGENI DETECTION IN DOG SERA FROM PATROCÍNIO, MG, BRAZIL ALTA FREQUÊNCIA DO SOROVAR COPENHAGENI DETECTADO EM CÃES DO MUNICÍPIO DE PATROCÍNIO, MG, BRASIL

This study aimed to evaluate the occurrence of canine leptospirosis and the possible risk factors associated with the disease in the municipality of Patrocínio, MG, Brazil. A cross-sectional observational study was carried out from July through August 2017. The municipality was divided into four regions (north, south, east and west) and a predefined number of neighborhoods (25) were randomly sampled in each region. Samples of blood serum were collected from 241 domiciled male and female dogs of different breeds and ages. To investigate the risk factors for canine leptospirosis, the owners of the animals were asked to fill out an epidemiological questionnaire. The following factors were evaluated: breed, sex, age, presence of rodents, type of diet, access to the street, vaccination, presence of flooded areas, and educational level of the owners. Blood serum samples were evaluated by the Microscopic Agglutination Test (MAT), using a collection of 24 live antigens. Of the 241 dogs evaluated, 32 (13.2%) were reactive. The most frequent serovars were: Copenhageni (37.5%) and Canicola (21.8%), followed by Icterohaemorrhagiae and Grippotyphosa (12.5%), Pomona, Tarassovi and Butembo (9.3%) and Hardjo (6.2%). The presence of canine leptospirosis was associated with purebred dogs (OR=0.3059 [95% CI: 0.1430 – 0.6547]) and vaccination (OR=2.581 [95% CI: 1.198 – 5.563]). It was concluded that some dogs in the municipality of Patrocínio, MG have anti-Leptospira spp. antibodies and that the serovars most frequently identified were Copenhageni (37.5%) and Canicola (21.8%). Pure breeds and vaccination were factors associated with the prevalence of infection.


INTRODUCTION
Leptospirosis is an infectious and zoonotic disease often occurring in developing countries. The disease is caused by the bacterium Leptospira interrogans, a spirochete-like etiological agent that comprises 22 different species, Ten of these species belong to group I (Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira borgpetersenii, Leptospira alexanderi, Leptospira weilii, Leptospira santarosai, Leptospira kmetyi, Leptospira alstoni and Leptospira mayottensis), five to group II (Leptospira licerasiae, Leptospira wolffii, Leptospira fainei, Leptospira broomii and Leptospira inadai) and seven to group III (Leptospira idonii, Leptospira meyeri, Leptospira terpstrae, Leptospira biflexa, Leptospira vanthielii, Leptospira yanagawa and Leptospira wolbachii). This disease affects mainly the renal proximal tubules, causing the patient to become a chronic renal carrier who eliminates the bacterium through his urine (ADLER; MOCTEZUMA, 2010). Leptospirosis can be transmitted in two ways: through indirect contact with contaminated soil, water and food, or through direct contact with infected animals (ACHA; SZYFRES, 2001). Rats are an important source of infection for dogs, since their habit of sniffing around garbage dumps and construction debris put them in contact with rat urine (NÓBREGA, 2015). On the other hand, dogs are part of the daily life of humans, and are thus an important source of infection for humans, since these animals can be renal carriers of the disease, even if they are asymptomatic (BIRCHARD;SHERDING, 2008). According to Brasil (2016), zoonotic diseases should be prevented through health education, environmental management to control and eliminate reservoirs, and through animal vaccination. The aim of this study was to evaluate the seroprevalence of canine leptospirosis and the risk factors associated with this infection in the LEMOS et al. Biosci The total population of dogs in the municipality of Patrocínio (MG) was estimated based on the human population of 88.648, according to the Brazilian Institute of Geography and Statistics for the year 2015. The ratio of dogs to humans was calculated based on a proportion of 1:6 (THRUSFIELD, 2004), which resulted in a total of 14,774 animals. The sample was calculated within a confidence interval of 95% and a variation of 5%. Recent surveys of canine leptospirosis in Brazil have revealed a prevalence ranging from 6.8% to 35.1% (FERNANDES et al., 2013;; therefore, an average prevalence rate of 20% was expected, resulting in a total of 241 samples. The animals of this study were male and female dogs of different breeds and ages, which appeared to be healthy. The blood samples were collected randomly in 25 neighborhoods of the municipality. The number of samples per neighborhood was estimated according to the number of dogs vaccinated per neighborhood during the 2016 Dog Rabies Vaccination Campaign, based on data provided by the municipality's Zoonosis Control Center (ZCC).
Blood samples were collected by venipuncture of the accessory cephalic using 3 mL syringes and a 25x7 needles. The samples were placed in tubes without anticoagulant and stored under refrigeration (2-8ºC), after which they were centrifuged at 2500 x g to obtain blood serum. The dogs' owners answered a questionnaire to determine the risk factors associated with canine leptospirosis. The following variables were investigated: breed, sex, age, access to the street, presence of rodents, existence of vacant lots near the home, the animal's diet, occurrence of floods, history of vaccination against leptospirosis, and the owners' level of education.
To calculate the frequency, the ratio of seroreactive dogs to the total number of sampled animals was established based on absolute and relative frequency, using descriptive statistical analysis.
The association between the epidemiological variables and positive animals was determined by means of the Odds Ratio nonparametric test for two independent samples, at a significance level of 5%, using GraphPad version 5.0 for Windows (San Diego, California, USA).
No.: number of reactive animals, * Animals reactive to more than one serovar with the same titration for both.
The prevalence rate found in this study (13.2%) was similar to the 13.1% found by Magalhães et al. (2007) in the city of Belo Horizonte, MG and lower than the 28.3% reported by Castro et al. (2015) in the city of Uberlândia, MG. Similar frequencies have been reported in other Brazilian states, such as 17.15% in Pará (PAZ et al., 2015), 19.7% in Paraíba (AZEVEDO et al. 2011) and 6.8% in Rio Grande do Norte (FERNANDES et al., 2013).
Most of the animals were reactive to the serovar Copenhageni (37.5%). Langoni et al. (2015) also reported this serovar as the one with the highest prevalence, while Fernandes et al. (2013) reported this serovar as the third most frequent. According to Mascolli et al. (2002), when the serovar Copenhageni is found to be the most prevalent, this indicates the influence of rodents, especially rats, as disseminators of the disease, since these animals are the main reservoirs of the disease. However, the presence of rodents was not identified as a risk factor for the occurrence of the infection (P=0.2966) in this study.
The serovar Canicola (21.8%) was the second most frequent. Castro et al. (2011) reported a similar finding in a study carried out in the city of Uberlândia, MG. Paz et al. (2015), Coiro et al. (2011) and Magalhães et al. (2007) also reported that the serovar Canicola appeared with the highest frequency. This serovar, whose natural hosts are dogs but which is also found in commercially available vaccines, causes the animal to present moderate clinical symptoms, but with a marked condition of leptospiremia, making it an important source of infection for humans (HAGIWARA et al., 2004). In dogs, the renal carrier state is more pronounced in the presence of the serovar Canicola than the serovars found in the serogroup Icterohaemorrhagiae (MAGALHÃES et al., 2006  Purebred animals presented a higher chance (OR=0.3059 [95% CI: 0.1430 -0.6547]) of infection than mixed breed dogs. This finding contradicts that of Nóbrega (2015) for purebred dogs. A higher frequency of anti-Leptospira spp. antibodies was found among dogs of the Shih Tzu breed. This may be attributed to the fact that small dog breeds are more popular today. Of the 77 purebreds evaluated here, only 17 were large dog breeds. Moreover, another factor possibly contributing to this finding is that these animals are often treated more carefully with respect to prevention measures, triggering vaccine reactions. Of the 18 purebred dogs that presented anti-Leptospira spp. antibodies, 15 had already been vaccinated. Two dogs tested positive for serovars Icterohaemorrhagiae and Copenhageni, which are part of the same serogroup Icterohaemorrhagiae. According to Costa (1996), this classification is based on their antigenic relationship, and their antigenic similarity enables the occurrence of crossreactions. One of the two aforementioned dogs had not been vaccinated against leptospirosis, while the other one was vaccinated 10 months before its blood was sampled.
Vaccination history must be taken into account when interpreting results (OIE, 2014). Vaccines for leptospirosis provide immunity ranging from three to 12 months (RODRIGUES et al., 2013). Of all the animals that showed anti-Leptospira spp. antibodies, six had been vaccinated less than 12 months before their blood samples were collected. Among them, four had titers of 100, one had a titer of 200 for serovar Pomona, and one had a titer of 3,200 for serovar Butembo.
Titers of 100 can be interpreted as evidence of previous exposure to Leptospira interrogans or of vaccine reaction (OIE, 2014).
In this study, vaccinated animals had a higher chance of having the infection (P=0.0200), but when compared to the variable of time elapsed from last vaccination, the latter showed no influence (P=1.0000). An absence of association can be explained by the fact that commercially available vaccines normally contain four serovars of the bacterium, i.e., Icterohaemorrhagiae, Canicola, Grippotyphosa and Pomona (TAVARES, 2015). Some vaccines also contain the serovars Copenhageni and Bratislava (FURQUIM, 2016), suggesting that vaccinated animals have not developed immunity or that the test was carried out within the vaccine's duration of protection, indicating that the positive result for anti-Leptospira spp. antibodies was due to vaccination.
The occurrence of leptospirosis was not associated with diet (P=0.5367), age (P=0.2174), sex (P=0.5692), level of education of the owners (P=0.2494), access to the street (P=0.2773), presence or absence of vacant lots near the residences (P=0.5706), and presence of rodents (P=0.2966).
It is concluded that dogs in the municipality of Patrocínio, MG, have anti-Leptospira spp. antibodies, and that the serovars Copenhageni (37.5%) and Canicola (21.8%) occurred most frequently. The risk factors associated with the prevalence of infection were the variables of being purebred and being vaccinated.