Study on coinfecting vector-borne pathogens in dogs and ticks in Rio Grande do Norte , Brazil

Since dogs presenting several vector borne diseases can show none or nonspecific clinical signs depending on the phase of infection, the assessment of the particular agents involved is mandatory. The present study aimed to investigate the presence of Babesia spp., Ehrlichia spp., Anaplasma spp., Hepatozoon spp. and Leishmania spp. in blood samples and ticks, collected from two dogs from Rio Grande do Norte showing suggestive tick-borne disease by using molecular techniques. DNA of E. canis, H. canis and L. infantum were detected in blood samples and R. sanguineus ticks collected from dogs. Among all samples analyzed, two showed the presence of multiple infections with E. canis, H. canis and L. infantum chagasi. Here we highlighted the need for molecular differential diagnosis in dogs showing nonspecific clinical signs.


Introduction
Ticks are important vectors of pathogens that may affect both animals and humans, causing morbidity and mortality in infected hosts (PAROLA; RAOULT, 2001).Dogs and humans are susceptible to infection by tick-borne agents, which include bacteria, protozoa and viruses.Recently, an increased risk of exposure to tick-borne pathogens among dogs has been observed around the world.Because of the close relationship that these pathogens have to humans, they have become a public health concern (BEUGNET;MARIÉ, 2009).
Among tick-borne diseases affecting dogs, canine monocytic ehrlichiosis (CME) is the most widespread illness reported in Brazil (VIEIRA et al., 2011).It is caused by Ehrlichia canis, an agent belonging to Anaplasmataceae that mainly parasitizes monocytes and is transmitted by Rhipicephalus sanguineus (DUMLER et al., 2001).Canine anaplasmosis is caused by Anaplasma platys and Anaplasma phagocytophilum, which infect dogs' platelets and neutrophils, respectively (DUMLER et al., 2001).Recently, A. phagocytophilum DNA has been detected in blood samples from dogs and in Amblyomma cajennense and R. sanguineus ticks in Rio de Janeiro (SANTOS et al., 2013).A. platys has been molecularly detected in dogs in the states of Mato Grosso do Sul, Paraná, São Paulo and Recife (SOUSA et al., 2013;SILVA et al., 2012;DAGNONE et al., 2009;RAMOS et al., 2010).Although R. sanguineus is a potential vector for A. platys (INOKUMA et al., 2000), the tick species involved in transmission of A. phagocytophilum in Brazil is still unknown.
Regarding babesiosis, apart from a single report of Babesia gibsoni in a dog from the state of Paraná (TRAPP et al., 2006), dogs are more often affected by Babesia vogeli in Brazil (PASSOS et al., 2005;FURUTA et al., 2009;SOUSA et al., 2013).
Leishmania parasites are predominantly transmitted by Lutzomyia spp.while feeding on blood.Although recent studies have incriminated ticks as suspected vectors of leishmaniasis (DANTAS-TORRES et al., 2010;COLOMBO et al., 2011), the real role of these arthropods in the transmission of Leishmania spp. is still unknown.
Since dogs presenting several vector-borne diseases may only show nonspecific clinical signs, or no signs at all, depending on the phase of infection, it is essential to assess the particular agents involved, given that some of these pathogens (especially A. phagocytophilum and Leishmania spp.) present potential threats to public health.
The present study aimed to investigate the presence of Babesia spp., Ehrlichia spp., Anaplasma spp., Hepatozoon spp.and Leishmania spp. in blood samples and ticks collected from two dogs in Rio Grande do Norte that showed signs suggestive of tick-borne disease, by using molecular techniques.

Blood samples and ticks collected
Blood samples and ticks were collected from two dogs showing clinical signs of vector-borne diseases that were attended at the teaching hospital of the Federal Rural University of the Semi-Arid Zone (UFERSA), Mossoró, Rio Grande do Norte, Brazil, in February 2013.
A two-month-old mixed-breed male dog (Dog #1) showed pale mucous membranes, apathy, dry hair, hepatosplenomegaly and presence of ticks on physical examination.This animal had a history of anorexia, vomiting and diarrhea.Although the owner reported having had the animal dewormed, the history of vaccination was not reported.According to the owner during anamnesis, there were no other animals in the same house where the sampled dog was kept.The dog had been acquired 20 days before the date of presentation to the teaching hospital, from a locality in the rural area of the municipality of Mossoró.Hematological and biochemical analyses showed the presence of hypochromic normocytic anemia, anisocytosis, thrombocytopenia, hyperproteinemia and elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
A three-year-old mixed-breed female dog (Dog #2) showed pale mucous membranes, dry thinning hair, onychogryphosis, scaly skin and crusts, lymphadenomegaly, hepatosplenomegaly and ocular mucous secretion on physical examination.This animal had had a history of hyporexia for approximately 2-3 months, with marked anorexia over the past 3 days, and apathy.According to the owner during anamnesis, although the sampled dog had outdoor access, there were no other animals in the same house where it was kept.Also, although the owner reported that the dog had been vaccinated against rabies, there was no history of deworming.Hematological analysis showed the presence of hypochromic normocytic anemia, anisocytosis, polychromasia, neutropenia, lymphopenia and thrombocytopenia.

Ticks
A total of seven ticks (three male and one female R. sanguineus ticks from Dog #1; one male and two female R. sanguineus ticks from Dog #2) were collected from the sampled dogs.The ticks collected were placed in tubes with 70% ethanol and stored at room temperature.After morphological identification as R. sanguineus (WALKER et al., 2000), the ticks were subjected to DNA extraction.

DNA Extraction
DNA was extracted from the dogs' blood samples and from individual ticks using the QIAamp DNA Blood and Tissue Mini Kit (QIAGEN, Valencia, California, USA), in accordance with the manufacturer's instructions.

Sequencing of PCR products
The reaction products were purified using the Silica Bead DNA Gel Extraction Kit (Fermentas®, São Paulo, SP, Brazil).The purified amplified DNA fragments were submitted for sequence confirmation in an automated sequencer (ABI Prism 310 Genetic Analyzer; Applied Biosystems/Perkin Elmer), in house, and were used for subsequent phylogenetic analysis.Consensus sequences were obtained through analysis on the sense and antisense sequences using the CAP3 program (http://mobyle.pasteur.fr/cgibin/MobylePortal/portal.py).Comparisons with sequences deposited in GenBank were conducted using the basic local alignment search tool (BLAST) (ALTSCHUL et al., 1990).The DNA sequences obtained in the present study were deposited in the GenBank database.

Results
DNA of E. canis, H. canis and L. infantum was detected in the blood samples and R. sanguineus ticks collected from these two dogs with nonspecific clinical signs of tick-borne diseases (Table 1).Both of the dogs were coinfected with at least two pathogens.The R. sanguineus ticks collected from both dogs were positive for at least one agent investigated.Among the nine samples analyzed, two of them (Dog #2 and R. sanguineus male 1") showed the presence of multiple infections with E. canis, H. canis, L. infantum.DNA of Babesia spp.and Anaplasma spp. was not detected in any of the blood and tick samples analyzed.The percentage identicalness and GenBank accession numbers of the DNA sequences amplified from dogs and ticks in the present study are shown in Table 2.

Discussion
In the present study, we reported on the existence of coinfection by vector-transmitted pathogens in blood samples and ticks collected from dogs showing clinical signs suggestive of arthropodborne diseases.To the authors' knowledge, this is the first report of simultaneous coinfection in dogs and ticks (R. sanguineus) by L. infantum, E. canis and H. canis.In addition, this study shows the first molecular detection of single or multiple infection by L. infantum, E. canis and H. canis in dogs and ticks in the state of Rio Grande do Norte, Brazil.
Recently, multiple vector-borne pathogen infection in dogs showing nonspecific clinical signs has been reported around the world.In the Caribbean region, Kelly et al. (2013)  Although Anaplasma spp.and Babesia spp.were not molecularly detected in dogs in the present study, these agents have previously been detected in dogs in Brazil.For instance, A. phagocytophilum has been detected in dogs and ticks, namely in R. sanguineus and A. cajennense, in the state of Rio de Janeiro (SANTOS et al., 2013).On the other hand, A. platys has been detected in dogs in the states of Mato Grosso do Sul, Paraná, São Paulo and Recife (SOUSA et al., 2013;SILVA et al., 2012;DAGNONE et al., 2009;RAMOS et al., 2010).B. vogeli is a widespread tickborne hemoprotozoon reported in Brazil (DANTAS-TORRES; FIGUEREDO, 2006).
The occurrence and distribution of these pathogens and their respective diseases in both animals and humans can be correlated with the geographical dispersion of ticks and other arthropod vectors (TROTTA et al., 2012).Multiple pathogens showing zoonotic potential have also been detected in the ticks Ixodes ricinus (A.phagocytophilum, L. infantum and Bartonella henselae) and R. sanguineus (Rickettsia conorii, R. massiliae, L. infantum and A. phagocytophilum) collected from domestic dogs in Europe and South America (TROTTA et al., 2012;PODSIADLY et al., 2007;COLOMBO et al., 2011;SANTOS et al., 2013;SMITH et al., 2013).Dogs parasitized by multiple pathogen-infected ticks may have an unknown clinical outcome that depends on the host-parasite relationship.Studies on interactions between tick-borne agents and Leishmania parasites with regard to establishment and progression of the disease are much needed (SOUSA et al., 2013).
Previously, in the state of Rio Grande do Norte, antibodies to Leishmania spp.were detected in 28% (39/139) of dogs showing clinical signs of leishmaniasis or asymptomatic dogs that lived in the same area as seropositive dogs (MATOS et al., 2006).Moreover, inclusions suggestive of Ehrlichia spp.were detected in 6.5% (13/198) of dogs showing clinical signs suggestive of canine monocytic ehrlichiosis (MEDEIROS; LIMA, 2004).
Since dogs infected by Anaplasma spp., E. canis, Leishmania spp., H. canis and/or Babesia spp.show nonspecific clinical signs (fever, weight loss, lethargy, splenomegaly, pale mucous membranes, vomiting and anorexia) and hematological abnormalities (anemia, leukopenia and thrombocytopenia) (CARDOSO et al., 2010;KELLY et al., 2013), a differential diagnosis based on identification of the etiological agents is important, in order to assess the zoonotic potential and the best therapy for the pathogens involved.
Since serological assays show cross-reactions (such as those found between E. canis and E. chaffeensis; A. platys and A. phagocytophilum; and Leishmania spp.and Trypanosoma cruzi) and direct detection of these pathogens by means of blood smears shows low sensitivity and specificity, especially in cases of chronic infection or low parasitemia, molecular techniques play a role as an important tool for detection and differentiation of pathogens that infect both animals and humans (LITTLE, 2010;LUCIANO et al., 2009).

Conclusion
The present work showed the presence of coinfection by multiple arthropod-borne pathogens (L.infantum, E. canis and H. canis) in dogs in Mossoró, state of Rio Grande do Norte, and highlighted the need for molecular differential diagnoses among dogs showing nonspecific clinical signs.

Table 2 .
Identity and GenBank accession numbers of sequences amplified from dogs and ticks in the present study, RN, Brazil.' ) ticks collected in Dog#1; ( " ) ticks collected in Dog#2; a Accession numbers deposited in Genbank from this study; Sample (R. s. male 1") was not sequenced for Ehrlichia spp. (