Ehrlichia canis and Rickettsia spp . in dogs from urban areas in Paraiba state , northeastern Brazil

The aims of our study was to identify Ehrlichia canis and antibodies against Rickettsia spp. belonging to the spotted fever group (SFG) in dogs sampled from Paraiba state, northeastern Brazil. Blood and serum samples collected by convenience from dogs in urban areas of five municipalities were analyzed by real-time PCR for the detection of E. canis DNA and by immunofluorescence assay test (IFAT) for the identification of antibodies against Rickettsia rickettsii, R. felis, R. parkeri, R. amblyommii and R. rhipicephali antigens. E. canis DNA was detected in 8.9% (64/719) of the blood samples, whereas 5.63% (43/763) of the serum samples were positive for at least one of the Rickettsia antigens tested by IFAT. This study showed for the first time the occurrence of E. canis and suggested the circulation of SFG Rickettsia in dogs in the study region of Paraiba state, northeastern Brazil.


Introduction
Rickettsioses are vector-borne diseases caused by bacteria of the Rickettsiales order, which include two families of obligate intracellular pathogens, Rickettsiaceae and Anaplasmataceae (DUMLER et al., 2001).Rickettsial infections are widely distributed and are transmitted by a variety of species of ticks.Thus, the epidemiology of several rickettsial infections is determined by the specific geographic distribution of the tick vector (DANTAS-TORRES, 2007;PAROLA et al., 2013).
Among members of the family Anaplasmataceae, Ehrlichia canis is the agent of canine monocytic ehrlichiosis (CME) in Brazil.CME is a multisystem disease manifesting as either acute, sub-clinical or chronic forms, depending on the virulence of the E. canis strain and the occurrence of co-infections, especially with other arthropod-borne pathogens, such as Babesia vogeli and Hepatozoon canis (VIEIRA et al., 2011).The diagnosis of CME is based on the observation of the presence of morulae within leucocytes in blood smears, isolation of the bacterium in cell culture, immunofluorescence assay test (IFAT), and molecular detection (PCR) (ROTONDANO et al., 2012).
Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, a member of the Spotted Fever Group (SFG), has long been recognized in Brazil, mainly in the southern and southeastern regions (ANGERAMI et al., 2009;PAROLA et al., 2013).More recently, the occurrence of other human pathogenic Rickettsia species such as R. felis, R. parkeri and Rickettsia spp.strain Atlantic rainforest has been increasingly reported in Brazil (HORTA et al., 2006;SILVEIRA et al., 2007;SPOLIDORIO et al., 2010).While R. felis was detected in Rhipicephalus sanguineus ticks in the state of Paraiba, northeastern Brazil (TANIKAWA et al., 2013), there was a recent report of a R. parkeri-like strain infecting Amblyomma nodosum ticks collected from birds in Paraíba (LUGARINI et al., 2015).The IFAT is the most common test and serological gold standard for the diagnosis of rickettsial infection in humans and animals (DANTAS-TORRES, 2007).
The knowledge of the occurrence of tick borne-agents among canine populations is crucial for early diagnosis, treatment and control of these diseases, preventing their spread to humans and other canine populations.This study aimed to investigate the occurrence of E. canis DNA and Rickettsia spp.antibodies in urban dogs in the state of Paraiba, northeastern Brazil.

Detection of Ehrlichia canis DNA by real-time PCR-DNA extraction and amplification
DNA was extracted from whole blood samples using a commercial DNA extraction kit (Wizard kit for DNA extraction  , Promega) (ROTONDANO et al., 2012).The detection of E. canis was performed by real-time PCR to amplify 378 base pairs (bp) of the dsb gene encoding a disulfide bond formation protein, using the primers Dsb321 and Dsb671 and a species-specific probe, as previously described (DOYLE et al., 2005).Positive and negative DNA samples were extracted from E. canis cultured in DH82 cells and uninfected cultures of DH82 cells, respectively.Controls were included for all PCR assays.
Briefly, serum samples were serially diluted in phosphate-buffered saline (PBS) in two fold dilutions from 1/64 up to 1/1280, and instilled on glass slides coated with the antigens.A commercial fluorescein, isothiocyanate-labelled rabbit anti-dog IgG (Sigma, St Louis, MO, USA), was used as secondary antibody.In each slide, a known non-reactive (negative control for all tested antigens) and a known reactive serum (positive control for all tested antigens) from the work of Krawczak et al. (2016) were tested in a 1/64 dilution.For each serum, the endpoint titer reacting with each Rickettsia antigen was determined, and the results were categorized as follows per Labruna et al. (2007): serum showing for a Rickettsia species titer at least four-fold higher than that observed for any other Rickettsia species was considered homologous to the first Rickettsia species or to a very closely related genotype.

Statistical analysis
A chi-square test was used to compare the prevalence of the pathogens and the geographic origin of the dogs analyzed.A p value <0.05 was considered statistically significant.All analyses were performed using the SPSS program version 13.0 for Windows.

Ethical considerations
This study was approved by the Ethics Committee of the Federal University of Campina Grande (PB) protocol number 07/2012.

Detection of E. canis DNA by real-time PCR
Ehrlichia canis DNA was amplified in 8.9% (64/719) of the dog blood samples analyzed by real-time PCR.The distribution of positive samples and the percentage of positivity per municipality are shown in Table 1.

Detection of anti-Rickettsia spp.antibodies by IFAT
Among the 763 dog serum samples analyzed by IFAT 5.63% (43/763) were reactive to at least one of the five rickettsial antigens tested, with titers ≥64.The titers of positive sera for the different species ranged from 64 to 512 for R. rickettsii and R. felis, 128 to 2048 for R. amblyommii, and 64 to 1024 for R. parkeri and R. rhipicephali.Three animals showed homologous reactions: one for R. amblyommii, one for R. felis, and one for R. rickettsii, which was four-fold higher than those for the other antigens tested (Table 2).
Only 0.55% (4/719) of the samples were positive for both agents tested in the present study.Four samples from Campina Grande municipality showed antibodies against at least one Rickettsia spp.antigens tested and had also DNA of E. canis.

Discussion
The present study assessed the occurrence of E. canis and exposure to other rickettsial agents in dogs from urban areas in the state of Paraiba in northeastern Brazil.Of the blood samples analyzed by real-time PCR, 8.9% yielded E. canis DNA.These results reveal a comprehensive picture of the CME situation in the region since real-time PCR reveals not only the exposure to the pathogen but also the infection process as ehrlichial DNA was detected.Even though Rotondano et al. (2015) had detected DNA of E. canis by real-time PCR in 25% (25/100) of the dogs sampled in Paraiba state, their samples comprised a veterinary hospital population, in contrast to random health dogs in the present study.This fact could be one of the reasons for the higher frequency of infected dogs in the study of Rotondano et al. (2015).
Here, we found 5.63% (43/763) of the canine serum samples reactive to at least one of the Rickettsia antigens.According to Piranda et al. (2008) the presence of seropositive animals indicates the circulation of Rickettsia of the SFG in an area at least 6-18 months earlier.As common hosts of tick-vectors for BSF, dogs are an important marker for disease in surveillance studies, and the occurrence of serologically positive dogs in a geographical area indicates the threat of human infection (ARAES-SANTOS et al., 2015).
This study provides evidence for exposure to Rickettsia spp. in urban dogs in the state of Paraiba.In the serum samples from three animals, it was possible to determine the probable antigen involved in homologous reaction (PAIHR) -R.amblyommii, R. felis and R. rickettsii.Among the other reactive animals, it was not possible to discriminate the infecting agent (Table 2).R. rickettsii is a known canine pathogen, whereas the role of pathogenicity by R. amblyommii in dogs is limited to serological evidence only.So far, the only tick-transmitted agents involved in human disease in Brazil are R. rickettsii and Rickettsia sp.strain Atlantic rainforest (ANGERAMI et al., 2009;SPOLIDORIO et al., 2010).The role of R. amblyommii in pathogenicity is not yet fully elucidated (BREITSCHWERDT et al., 1988).However, some Rocky Mountain Spotted Fever (RMSF) cases in the U.S. that were attributed to R. rickettsii could be due to R. amblyommii instead (APPERSON et al., 2008).
In Brazil, two species of ticks are implicated in transmission of R. rickettsii to humans and other animals, Amblyomma sculptum (published as A. cajennense) and A. aureolatum (KRAWCZAK et al., 2014;SARAIVA et al., 2014).While A. aureolatum is not present in northeastern Brazil (SARAIVA et al., 2014), A. sculptum is a very rare or absent tick species in the Caatinga biome of northeastern Brazil (MARTINS et al., 2016).Therefore, it is unlikely that A. sculptum acts as an important vector of R. rickettsii within this biome.
R. sanguineus is the main ectoparasite in dogs in the dry Agreste and Sertão mesoregions of the Paraiba state (ROTONDANO et al., 2015).Although Tanikawa et al. (2013) had detected R. felis DNA in 4,5% (1/22) of R. sanguineus collected from Paraiba state, the role of R. sanguineus as vector of R. rickettsii in Brazilis not totally elucidated, further studies are needed to confirm its role as a vector among dogs in state of Paraiba.
In spite of the fact that Ctenocephalides felis, a host for R. felis, is the most common flea species infesting dogs in Brazil (OLIVEIRA et al., 2002), only three animals of the present study displayed R. felis antibodies and one of them showed antibody against R. felis four-fold higher than that observed for any other Rickettsia species antigens, being thus considered homologous to R. felis.The potential presence of R. felis in dogs in the study region has important implications since R. felis has already been considered as a human pathogen (SCHRIEFER et al., 1994;LABRUNA et al., 2007).
A recent study detected two Rickettsia species infecting bird ticks in the Atlantic forest of Paraíba state: Rickettsia sp.strain NOD (a R. parkeri-like agent) in Amblyomma nodosum, and R. amblyommii in A. longirostre (LUGARINI et al., 2015).Since these two ticks species have occasionally been reported infesting dogs in Brazil (MORAES-FILHO et al., 2009;SABATINI et al. 2010), it is possible that they could be involved in the seropositivity of some of the dogs in the present study; however, we have no data on the history of tick infestations of our canine sample.

Conclusions
This study is the first to detect both antibodies to SFG Rickettsia and DNA of E. canis in dogs from the state of Paraiba in northeastern Brazil.The finding of infection by E. canis in the whole blood of the animals analyzed reveals the endemic status of CME in the region.The study detected, for the first time, a positive serological reaction indicating Rickettsia spp.antibodies in forty-three samples, and the probable antigen involved in a homologous reaction (R. amblyommii, R. felis and R. rickettsii) was identified in three samples.Taking into account that the dogs can serve as important indicators of tick-borne diseases, further studies to better understand the dynamics of rickettsiae diseases and their vectors in this region are extremely important to know about the real situation the circulation the rickettsias in the Paraiba state, northeastern Brazil.

Figure 1 .
Figure 1.Geographical location of the municipalities included in the study of Ehrlichia canis and Rickettsia spp. in dogs from urban areas in Paraiba state, northeastern Brazil.

Table 1 .
Distribution by municipality of the number of dogs from Paraiba, Brazil, analyzed for Ehrlichia canis DNA by real-time PCR and Rickettsia spp.by indirect immunofluorescence assay test (IFAT) showing number of positives and percentages.

Table 2 .
Distribution by municipality of the number of dogs from Paraiba state, Brazil, analyzed for Rickettsia spp.by indirect immunofluorescence assay test (IFAT) showing the number of positives and percentages.
PAIHR: probable antigen involved in homologous reaction.