Serosurvey of Tick-borne Pathogens in Dogs from Urban and Rural Areas from Parana State, Brazil

Avaliação sorológica de patógenos transmitidos por carrapatos em cães urbanos e rurais do estado do Paraná, Brasil Abstract Considering the zoonotic potential of tick-borne disease (TBD) agents and the fact that dogs may act as sentinels for human infection, the aim of the present study was to determine the seroprevalence of TBD agents and risk factors for exposure in two different canine populations from Parana State, Southern Brazil. A total of 138 dog serum samples from urban (UA) (n=68) and rural (RA) (n=70) areas were tested with commercial ELISA rapid test for Anaplasma phagocytophilum, Ehrlichia canis and Borrelia burgdorferi antibodies and indirect immunofluorescence assay (IFAT) for Babesia vogeli. An overall of 92/138 (66.7%) dogs, being 62/68 (91.2%) from UA and 30/70 (42.9%) from RA, were seropositive for at least one TBD agent. From the total number of dogs, sixty-two were positive for E. canis (44.9%), 19 (13.8%) for A. phagocytophilum, and 64 (46.4%) for B. vogeli. Anti-B. burgdorferi antibodies were not detected. Dogs from UA showed a higher percentage of tick infestation (p = 0.0135) and were highly associated with seropositivity to E. canis (p = 0.000005), A. phagocytophilum (p = 0.0001), and B. vogeli (p = 0.0012). In summary, the findings indicate that dogs from urban areas present higher potential risk exposure to TBD pathogens than those from rural areas. Resumo Considerando o potencial zoonótico das doenças transmitidas por carrapatos (DTCs) e que os cães podem atuar como sentinelas para infecções em humanos, os objetivos deste estudo foram determinar a soroprevalência de agentes das DTCs e fatores de risco para a exposição em duas diferentes populações caninas do Estado do Paraná, região Sul do Brasil. Um total de 138 amostras de soro de cães de área urbana (AU) (n = 68) e rural (AR) (n = 70) foram testadas utilizando um teste de ELISA comercial rápido para detecção de anticorpos contra Anaplasma phagocytophilum, Ehrlichia canis e Borrelia burgdorferi e imunofluorescência indireta (IFI) para Babesia vogeli. Um total de 92/138 (66,7%) cães, sendo 62/68 (91,2%) da AU e 30/70 (42,9%) da AR, foram soropositivos para pelo menos um agente. Do número total de amostras, sessenta e duas (44,9%) foram positivas para E. canis, 19 (13,8%) para A. phagocytophilum e 64 (46,4%) para B. canis vogeli. Anticorpos anti-B. burgdorferi não foram detectados. Os cães da AU apresentaram o maior percentual de infestação por carrapatos (p = 0,0135) e foram altamente associados …

It has been suggested that R. sanguineus, widely found across the country, may be a potential vector for Anaplasma platys to dogs in Brazil (DANTAS-TORRES, 2008).The identification of this organism can be achieved by the observation of A. platys inclusions in platelets during the examination of stained peripheral blood smears and by molecular methods (DAGNONE et al., 2009).Prevalence of A. platys infection ranges from 18.8% to 48.8% (DAGNONE et al., 2009;SANTOS et al., 2009;RAMOS et al., 2010); however, the seroprevalence remains to be fully established, since there is no developed serological diagnostic assay for this agent to date.Serological assays, such as ELISA, immunofluorescence antibody test (IFAT) and dot-ELISA, originally developed for the detection of anti-A.phagocytophilum antibodies, have been used as an alternative, due to the cross-reaction between A. phagocytophilum and A. platys antibodies (CHANDRASHEKAR et al., 2010).
A. phagocytophilum (a combination of organisms previously known as E. equi, E. phagocytophila and human granulocytic ehrlichiosis [HGE] agent) is the causative agent of granulocytic anaplasmosis and has been reported infecting dogs, cats, horses, ruminants and humans worldwide (WOLDEHIWET, 2010).Ixodes scapularis, I. pacificus and I. ricinus ticks act as the main vectors for this agent (AMUSATEGUI et al., 2008;BOWMAN et al., 2009) and although these ticks are not present in Brazil, other Ixodes and Amblyomma species are frequently found (FIGUEIREDO et al., 1999;ABEL et al., 2000).Recently, A. phagocytophilum was detected, by molecular methods, infecting dogs in Brazil (SANTOS et al., 2011).
Ticks of the Ixodes genus are also involved in the transmission of Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme disease (BURGDORFER et al., 1989).In Brazil, it is suspected that both Amblyomma cajennense and Rhipicephalus (Boophilus) microplus are involved in the transmission of B. burgdorferi (s.l.) (BARROS-BATTESTI et al., 2000;YOSHINARI et al., 2003).There are serological evidences of B. burgdorferi (s.l.) infection in Brazilian dogs, with prevalence data ranging from 0.04% to 53.5% (LABARTHE et al., 2003;CARLOS et al., 2007;SPOLIDORIO et al., 2010).Nevertheless, it is important to mention that B. burgdorferi (s.l.) has never been isolated or molecularly detected in this country, and thus, the role of these ticks on the transmission of Lyme disease remains to be established.
Considering the zoonotic potential of these agents and that dogs may act as sentinels for tick-borne infections to humans (AMUSATEGUI et al., 2008), the aims of the present study was to determine the seroprevalence of A. phagocytophilum, E. canis, B. burgdorferi and B. vogeli, the identification of tick species found in dogs, and the evaluation of the risk factors for exposure to the organisms, in two different canine populations from Paraná State, Southern Brazil.

Ethical principles
The study was approved by the Ethics Committee in Animal Experimentation and Animal Welfare of the "Universidade Estadual de Londrina" -UEL (number 34/2011).

Study area
The study was carried out in rural and urban areas located in the central-northern region of the State of Parana.The urban area (UA) is located in a region of 'Jardim California', a neighborhood located in the eastern side of Londrina (23° 08' 47" S and 51° 19' 11" W).The area is situated at 610 m above sea level; presents a subtropical climate with rainfall throughout the year, but concentrated during summer.The average annual temperature is around 20 °C (INMET, 2011).Dogs have free access to an abandoned square, covered by a mixed overgrowth pasture composed by grass and undesired plants (bushes, scrubs); dogs are also in constant contact with horses.This square is also used for neighbors' leisure, and present high amount of ticks.
The rural area (RA) is a settlement situated in the 'Alvorada do Sul' county (22° 54' 34.4" S and 51° 13' 49.1" W).The area is located within the rural perimeter of Alvorada do Sul, 16 km far from downtown, 380 m above sea level.The region presents subtropical climate with rainfall throughout the year, but with a tendency of concentrating rain during the summer months with an average temperature of 25 °C (INMET, 2011).The area is subdivided in 60 land properties with approximate area of 12 ha each, totalizing 786 ha.The rural settlement lacks basic sanitation and the main activity of dwellers is the cultivation of grains and vegetables.The area also comprises 20% of native forest, presenting diverse fauna, with a varied population of wild animals.In this region, a large number of ticks can be found throughout the year.

Animals
A total of 138 dogs of different ages, breeds, and both sexes from urban and rural areas of the central-northern region of the State of Parana were used.A total of 68 dogs from UA and 70 dogs from RA were sampled during an active surveillance program for infectious diseases.

Collection of ticks
A total of 126 adult ticks were collected from dogs, being 59 from the UA and 67 from the RA.Ticks were removed using tweezers and placed in tubes containing 70% ethanol solution and identified according to morphological keys (ARAGÃO; FONSECA, 1961;GUIMARÃES et al., 2001).

Study design
Owners responded to a previous validated epidemiological questionnaire during the sampling, addressing breed, age, gender and presence of ticks.Age was classified in groups: ≤ 1 and > 1 year old.

Sampling
Dog blood samples (10 mL) were collected by venipuncture of jugular vein into tubes without anti-coagulant and kept at room temperature (25 °C) until visible clot retraction, centrifuged at 1500 g for 5 minutes.Sera were separated and kept at -20 °C until testing.

Detection of antibodies against A. phagocytophilum, E. canis and B. burgdorferi (s.l.)
All 138 dogs serum samples were tested for A. phagocytophilum, B. burgdorferi (s.l.), and E. canis using a commercial ELISA rapid test (SNAP  4Dx  , IDEXX Laboratories Inc., Westbrook, ME, USA), according to the manufacturer's instructions.

Detection of antibodies against B. vogeli
Antibodies anti-B.vogeli were detected by indirect immunofluorescence antibody test (IFAT) using antigens obtained from a splenectomized dog inoculated with B. vogeli, as previously described (TRAPP et al., 2006), with modifications.Briefly, IFAT was performed with 10 µL of serum samples incubated at 37 °C for 30 minutes in slides previously seeded with B. vogeli and washed three times for 5 minutes in phosphate buffered saline (PBS, pH 7.2).Ten microliters of fluorescein isothiocyanateconjugated rabbit anti-dog IgG (Sigma-Aldrich, St. Louis, MO) at 1:1000 dilution in 0.01% Evans blue was applied onto the slide.Slides were then incubated at 37 °C for 30 minutes, washed three times for 5 minutes, allowed to air dry and, subsequently, examined in microscope with fluorescent light source.Serum samples with fluorescent protozoa at dilution ≥ 1:80 were considered positive.Titers were determined to the largest dilution in which fluorescence was visualized around the protozoa.

Statistical analysis
Either Chi-square or Fisher's exact test was used to determine the difference between whether individual factors were associated with seropositivity to A. phagocytophilum, B. burgdorferi (s.l.), E. canis and/or B. canis.Odds ratio (OR), 95% confidence interval and p values were calculated separately for each variable.Results were considered statistically significant when p < 0.05.Data were compiled and analyzed using Epi Info ™ Software (version 3.5.3).
Dogs living in the urban area were highly associated with seropositivity to E. canis (p = 0.000005), A. phagocytophilum (p = 0.0001), and B. vogeli (p = 0.0012).Dogs > 1 year old were highly associated with seropositivity to E. canis (p = 0.00001).No significant association was found between age or gender and seropositivity to A. phagocytophilum and B. vogeli.Results for the seroprevalence of A. phagocytophilum and E. canis in dogs from rural and urban areas within each variable studied are shown in Table 1.

Discussion
In Brazil, data on the distribution of arthropods and canine TBDs are still incomplete.In this study, 91.2% of the dogs from the urban area (UA) were seropositive for at least one tick-borne disease agent, and were 13.7 times more likely to be seropositive than those living in rural areas (RA).Besides, previous studies have shown a wide variation in seroprevalence for different TBD agents, such as E. canis, B. vogeli and B. burgdorferi, in different regions of Brazil (LABARTHE et al., 2003;CARLOS et al., 2007, SPOLIDORIO et al., 2010), but analyses were performed individually for each agent, and thus, there is no data to compare the overall seroprevalence found.
The higher seroprevalence found in the present study in dogs from UA was mainly due to seropositivity to E. canis (64.7%) and B. vogeli (60.3%), both agents transmitted by R. sanguineus ticks.Animals living in urban areas have a higher chance of becoming infested with R. sanguineus ticks than dogs dwelling rural areas (LABRUNA et al., 2001).This fact was supported by the present study, where 70% of the dogs from UA were infested by ticks, with 98.3% of these identified as R. sanguineus.High infestation rates of R. sanguineus have been shown to increase the risk of attack to humans (USPENSKY; IOFFE-USPENSKY, 2002).A previous study, also performed in the municipality of Londrina, showed that owners of tick-infested dogs were 3.2 times more likely to have removed ticks from themselves (TRAPP et al., 2006).Thus, there is a potential risk of human infection by TBD agents in the UA studied.
In this study, 60.3% of the dogs from UA were seropositive for B. vogeli.Lower seroprevalence data (35.7%)were obtained in a hospital population of dogs in the same study area (TRAPP et al., 2006).A previous study showed no difference in seroprevalence for B. canis between rural (46.5%) and urban (42.9%) areas in the State of Espirito Santo (SPOLIDORIO et al., 2010).In the present study, dogs from UA were 3.1 times more likely to be seropositive to B. vogeli than those from RA (p = 0.0012).However, association between age, sex, gender and seropositivity to B. vogeli was not observed (Table 1).The difference in seroprevalence found in other studies may have been due to climatic variation, population studied, diagnostic test used, and the cut-off used in the IFAT to B. vogeli.Dogs from UA were 5.29 times more likely to be seropositive to E. canis than dogs from RA (p < 0.05).Besides, higher percentage of tick infestation have been found in dogs from UA (70.6%) than those from RA (50%) (p = 0.0135), which may explain the seroprevalence difference; association between seropositivity to E. canis and the presence of ticks was not observed (p = 0.9192).However, the relatively small number of samples analyzed has limited statistical power and thus, differences might have been missed.Serological surveys of E. canis in dogs have found seroprevalence data ranging from 4.8% to 38% and from 24.7% to 65.6% in urban and rural areas, respectively, using different methods, such as IFAT and ELISA (VIEIRA et al., 2011).The commercial ELISA rapid test used in this study utilizes synthetic peptides from p30 and p30-1 outer membrane proteins of E. canis as antigen, and detects anti-E.canis and anti-E.chaffeensis antibodies (O'CONNOR et al., 2006; SNAP  4Dx  product insert and IDEXX Laboratories, unpublished data).In a previous study, the assay was able to identify only 30% of the serum samples from dogs with low-titer (80 to 160) on E. canis-IFA, but identified all dogs with titers > 320 (O'CONNOR et al., 2006).Thus, seroprevalence for E. canis in dogs from the studied areas may be higher, since dogs with low titers may not have been recognized when a point-of-care ELISA assay was used, as previously described in dogs from an animal shelter in central Spain (COUTO et al., 2010).Antibodies against A. phagocytophilum were found in 13.8% of the dogs using the commercial ELISA.Different results were found in dogs from the United States, France and Central Spain, which reported seroprevalence data of 4. 8%, 2.72%, and 19% (BOWMAN et al., 2009;PANTCHEV et al., 2009;COUTO et al., 2010), respectively.Although A. phagocytophilum infection was reported in dogs from the State of Rio de Janeiro, southeastern Brazil, by a quantitative real-time PCR followed by sequencing of the msp2 gene (SANTOS et al., 2011), no serological data regarding this agent have been reported for Brazilian dogs to date.It is worth mentioning that the results found in the present study may be due to the cross-reactivity between A. phagocytophilum and A. platys in the assay used (CHANDRASHEKAR et al., 2010; SNAP  4Dx  product insert and IDEXX Laboratories, unpublished data).Using the same commercial ELISA rapid test in serum samples from 16 PCR-positive to A. platys dogs, in acute stage of disease, cross-reaction between these two bacteria was not found (FERREIRA et al., 2008).Unfortunately, PCR was not performed in the present study and it was not possible to establish whether antibodies detected were due to A. phagocytophilum or A. platys.Further studies should be conducted in order to elucidate the cross-reactivity between A. phagocytophilum and A. platys.

Conclusion
From two different populations, it was possible to determine seroprevalence data of two major tick-borne disease agents, E. canis and B. vogeli, which cause severe clinical illness in dogs in Brazil.Moreover, the findings of this study indicate a higher potential risk for dogs exposure to TBD pathogens in urban than in rural areas.

Table 1 .
Serological prevalence of Anaplasma phagocytophilum, Ehrlichia canis, Babesia vogeli in dogs within each variable studied, State of Parana, southern Brazil.