Occurrence of Mycoplasma haemocanis in dogs infested by ticks in Campo Grande , Mato Grosso do Sul , Brazil Ocorrência de Mycoplasma haemocanis em cães infestados por carrapatos em Campo Grande , Mato Grosso do Sul , Brasil

Hemotropic mycoplasmas in dogs, such as Mycoplasma haemocanis, have been described worldwide. Recently, these pathogens have been reported to be causative agent of zoonosis. It is known that its transmission may occur through the action of blood-sucking arthropods (e.g. ticks or fleas), through blood transfusion, contaminated fomites and/or transplacentally. In Brazil, M. haemocanis is present in practically all regions and the tick Rhipicephalus sanguineus sensu lato is suspected the main vector. In the municipality of Campo Grande, state of Mato Grosso do Sul, there is little information about infection of dogs by M. haemocanis, or on the main epidemiological features associated with it. Thus, the aim of the present study was to determine the occurrence of M. haemocanis among dogs infested by ticks and to assess possible associations with some epidemiological factors. The polymerase chain reaction (PCR) and DNA sequencing were used to analyze dog blood samples (n = 94). DNA from M. haemocanis was detected in four samples. No significant associations were observed with any epidemiological parameter analyzed here. However, the results from this study confirm that this pathogen is circulating in this region and should be considered in the differential diagnosis of diseases among anemic dogs.


Introduction
The genus Mycoplasma comprises a group of bacteria without a cell wall and with a diminutive genome that makes them strictly dependent on the host cell.These bacteria may infect several species of animals and are associated with apathy, arthritis, fever, hemolytic anemia, infertility, lethargy, respiratory disorders, lymphadenopathy, pale mucosae, urogenital disease and weight loss (CHALKER, 2005;WILLI et al., 2007;COSTA, 2011;SILVA-SANTOS et al., 2014).
Most of infected dogs show chronic asymptomatic infection (HOSKINS, 1991;CHALKER, 2005;COSTA, 2011).In fact, disease manifestations in animals are most frequently reported in special cases, for example in association with drugs or retrovirus-induced immunosuppression, poor nutrition, pregnancy, lactation and other concomitant diseases (MACIEIRA et al., 2008).
In Brazil, canine hemotropic mycoplasmas has been reported in the northeastern region (RAMOS et al., 2010;SILVA-SANTOS et al., 2014), southeastern region (O'DWYER et al., 1997;ALVES et al., 2014), southern region (VALLE et al., 2014) and central-western regions (COSTA, 2011).In the state of Mato Grosso do Sul (MS), which is located in the central-western region of Brazil, only one survey of hemoplasmas mycoplasmas has been conducted so far, among cats (SANTIS et al., 2014).Therefore, the aim of the present study was to assess the occurrence of hemotropic mycoplasmas among dogs in Campo Grande, MS, Brazil, and to identify the main epidemiological features associated with the positivity to these pathogens.

Materials and Methods
The present study was approved by the Ethics Committee for Animal Experimentation of the Federal University of Mato Grosso do Sul (protocol number 592/2014), from August 2014 to April 2015.A convenience sampling was conducted by collecting 94 blood samples (n = 94) from dogs at the Zoonosis Control Center of the municipality of Campo Grande, state of Mato Grosso do Sul, Brazil.Samples were collected by puncturing cephalic vein and the material obtained was stored in sterile tubes containing EDTA (ethylenediaminetetraacetic acid).
Sampling was performed only from dogs infested by ticks, with no restrictions regarding gender, breed, age or origin.Clinical and epidemiological information on all animals was obtained (i.e.breed, gender, contact with fleas, outdoor access and urban or rural origin).Ticks were collected and stored in 70% ethanol for further identification using the identification key modified by Barros-Battesti et al. (2006).
DNA samples were extracted from 350µl of blood using the methodology described by Araújo et al. (2009).The concentration and integrity of the extracted samples were evaluated by means of spectrophotometry (BioPhotometer Plus; Eppendorf ® ) and agarose gel electrophoresis (1%), respectively.In order to check for the absence of PCR inhibitors, all samples were subjected to PCR targeting the β-actin gene (WANG et al., 2007).
Afterwards, the samples were analyzed through conventional PCR for Mycoplasma sp., using a primer set (fHf5 and rHf6) previously described to identify Mycoplasma haemofelis in cats (MESSICK et al., 1998) and later successfully used to detect M. haemocanis in a dog (BRINSON & MESSICK, 2001).
The PCR reactions were performed in a final volume of 25 µL, containing 10 mM Tris-HCL (pH 8.3), 50 mM of KCl, 1.5 mM MgCl 2 , 0.2 mM of each deoxynucleotide (dNTP), 1.25 U of Taq DNA polymerase (Ludwig Biotec), 11 pmol of each primer, and approximately 100 ηg of genomic DNA.The cycling condition were: 94 °C for three minutes, followed by 30 cycles at 94 °C for one minute, annealing at 51 °C for 30 seconds and extension at 72 °C for 40 seconds.A final extension step at 72 °C for three minutes was performed.
Mycoplasma haemocanis positive sample used as the positive control was obtained in a previous study, and its DNA sequence is deposited in GenBank under accession number FJ911910 (RAMOS et al., 2010).Nuclease-free water was used as the negative control.
The amplification products were viewed under ultraviolet light after electrophoresis on agarose gel (2%) stained with GelRed (Biotium) according to manufacturer's instructions.
Positive amplicon bands were cut from gels and purified using a commercial kit (Qiaex II; Qiagen), in accordance with the manufacturer's instructions.The samples were sequenced in both directions using an automated sequencer (3130 ABI; Applied Biosystems).The chromatograms were evaluated and edited using BioEdit v.7.2.5 software (HALL et al., 1999), and consensus sequences were subjected to BLASTn search (ALTSCHUL et al., 1990), in order to determine the sequence identity by comparison with orthologous sequences available in the GenBank database.

Results and Discussion
All the DNA samples showed amplification for the constitutive gene (β-actin), indicating thus the absence of PCR inhibitors.
Four (4.25%) out of the 94 blood samples analyzed revealed amplicons (650bp) compatible with Mycoplasma sp., in accordance with Messick et al. (1998).Through DNA sequence analysis, it was possible to identify the species Mycoplasma haemocanis (99% identicalness with sequences available in the GenBank database).Considering that all the DNA sequences obtained in the presented study showed 100% of identity with each other, only one sequence was deposited in GenBank, under the accession number KT163241.
A total of 345 ticks were collected from the animals sampled, and all were identified as R. sanguineus s.l.
Studies on frequency using PCR to detect M. haemocanis have been conducted in several regions of Brazil, and have presented similar results (RAMOS et al., 2010;COSTA, 2011;ALVES et al., 2014).In the present study, only samples from dogs parasitized by R. sanguineus s.l. were used.
In Brazil, despite the high rate of infestation by R. sanguineus s.l.among dogs (LABRUNA & PEREIRA, 2001;ALMEIDA et al., 2013), the frequency of hemotropic mycoplasmas is generally low, especially when compared with other tick-borne pathogens such as Ehrlichia canis, Anaplasma platys and Babesia vogeli, also transmitted by ticks (RAMOS et al., 2010;COSTA, 2011).This observation may raise questions about role of R. sanguineus s.l. as the main vector of Mycoplasma, especially because there is only one study that has proven the capacity of R. sanguineus in transmitting M. haemocanis (SENEVIRATNA et al., 1973).In fact, studies have correlated the frequency of infection and presence of ticks in dogs.However, significant associations have not always been observed (BARKER et al., 2010).Moreover, the action of other arthropod vectors should be considered, since the same climatic conditions that favor tick development may also favor them.Thus, when a positive correlation between ticks and hemoplasmas is found, probably it is also found for these other arthropods.In addition, other transmission routes in dogs, including the participation of other arthropods should be investigated.
Several arthropods (e.g.ticks, fleas, lice, mites, flies and mosquitoes) have been identified as vectors of numerous species of hemoplasmas in different vertebrate hosts (Table 1).Most of them are known to infest different species and represent a possible interspecific transmission vector, as probably occurred in animal-origin human mycoplasmosis (SANTOS et al., 2008;MAGGI et al., 2013).Therefore, it is mandatory to investigate the vector competence of other arthropods in relation to hemotropic mycoplasmas transmission to dogs, since the importance of the R. sanguineus tick remains unclear.
In the present study, was not possible to perform an association study between M. haemocanis PCR positive results and epidemiological parameters (i.e.breed, gender, contact with fleas, outdoor access and urban or rural area) due to the low number of PCR positive samples.However, no significant associations were found in studies carried out in France (KENNY et al., 2004), Switzerland (WENGI et al., 2008), and Brazil (ALVES et al., 2014).Although association between contact with fleas and infection by M. haemocanis was not evaluated in the present study, three of the four infected animals had a history of flea infestation.
Interestingly, in southern Europe, Novacco et al. (2010) observed that young mongrel dogs living in shelters were significantly more predisposed towards infection by hemoplasmas.On the other hand, in Tanzania and Trinidad and Tobago (BARKER et al., 2010), male dogs with free access to outdoor environments were significantly more likely to present infection by hemoplasmas.In these cases, contact with infected blood during fights would probably favor infection, since no association with tick infestation was observed.Although contact with saliva was not confirmed as a risk factor for infection, a study by Sazaki et al. (2008) showed that dogs of the Tosa breed, which are usually known as fighting dogs, are more prone to infection in Japan.Immunosuppression and weakness caused by other concomitant diseases have been identified as important factors that favor infection by these agents (PRYOR & BRADBURY, 1975;KRAKOWKA, 1977;GRETILLAT, 1981;NOVACCO et al., 2010;ROURA et al., 2010).In Brazil, coinfections by M. haemocanis and other tick-borne pathogens in dogs have also been reported (TRAPP et al., 2006;RAMOS et al., 2010).In the present study, two dogs were positive for M. haemocanis and sero positive for Leishmania sp., but it was not possible to assure the correlation between the pathogens (i.e.M. haemocanis and Leishmania sp.).Because of low sanitary conditions of these animals, other pathogens might also be present, but detection of microorganisms other than hemoplasmas was outside the scope of this study.
Despite the low frequency of M. haemocanis infection in dogs in Campo Grande (4/94), the results from this study confirm that this pathogen is circulating in this region and should be considered in the differential diagnosis of diseases among anemic dogs.

Table 1 .
Hemoplasma species and their known vectors for some vertebrate hosts.