Maia et al. Parasites & Vectors (2016) 9:170 DOI 10.1186/s13071-016-1452-2 SHORT REPORT Open Access Molecular detection of Leishmania infantum, filariae and Wolbachia spp. in dogs from southern Portugal Carla Maia1,2* , Laura Altet3, Lorena Serrano3, José Manuel Cristóvão1, Maria Dolores Tabar4, Olga Francino3,5, Luís Cardoso6, Lenea Campino1,7 and Xavier Roura8 Abstract Background: Leishmaniosis caused by the protozoan Leishmania infantum and dirofilariosis caused by the nematodes Dirofilaria immitis or Dirofilaria repens are vector-borne zoonoses widely present in the Mediterranean basin. In addition, some studies reported that the endosymbiont Wolbachia spp. play a role in the biology and pathogenesis of filarial parasites. The aim of this work was to evaluate the frequency of mono- and co-infections by L. infantum, filariae and Wolbachia spp. and their association with clinical signs in dogs from the south of Portugal. Leishmanial, filarial and Wolbachia spp. DNA were evaluated by specific real-time polymerase chain reaction (qPCR) assays in blood samples from 230 dogs. Findings: One hundred and thirty-nine (60.4 %) dogs were qPCR-positive for L. infantum and 26 (11.3 %) for filariae (24 for D. immitis only, one D. immitis and for Acanthocheilonema dracunculoides and another one for Acanthocheilonema reconditum only). Wolbachia spp. DNA was amplified from 16 (64.0 %) out of the 25 D. immitis-positive dogs. Nineteen (8.3 %) dogs were co-infected with L. infantum and D. immitis, including the one (0.4 %) A. drancunculoides-positive animal. In dogs without clinical signs consistent with leishmaniosis and/or dirofilariosis, L. infantum prevalence was 69 %, whereas in those dogs with at least one clinical manifestation compatible with any of the two parasitoses prevalence was 42.7 %. Leishmania prevalence was significantly higher in apparently healthy mongrels (77.2 %) and pets (76.9 %) than in defined-breed dogs (including crosses; 58.8 %) and in dogs with an aptitude other than pet (i.e. farm, guard, hunting, shepherd or stray), respectively, whereas in those dogs with at least one clinical sign, the detection of L. infantum DNA was higher in males (53.3 %) and in those dogs not receiving insect repellents (52.8 %). Conclusions: The molecular detection of canine vector-borne disease (CVBD) agents, some of which are zoonotic, reinforces the need to implement efficient prophylactic measures, such as insect repellents and macrocyclic lactones (including compliance to administration), in the geographical areas where these agents are distributed, with the view to prevent infection and disease among mammalian hosts including humans. Keywords: Dogs, Dirofilaria immitis, Leishmania infantum, Portugal, Wolbachia spp Findings central role in the transmission cycles of some vector- Canine vector-borne diseases (CVBD) constitute a diver- borne agents by acting as reservoirs and sentinels of sified group of illnesses, which are caused by a multitude human infections, thus making the control of CVBD de- of pathogens transmitted by arthropod vectors [1]. In sirable under the One Health umbrella. addition to their veterinary importance, dogs play a Leishmaniosis caused by the protozoan Leishmania infantum and heartworm disease and subcutaneous filar- * Correspondence: carlamaia@ihmt.unl.pt iosis respectively caused by the nematodes Dirofilaria 1 Global Health and Tropical Medicine, Medical Parasitology Unit, Instituto de immitis and Dirofilaria repens are three vector-borne zoo- Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal 2 Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e noses widely present in the Mediterranean basin, with Tecnologias, Lisbon, Portugal transmission of the first one by phlebotomine sand flies of Full list of author information is available at the end of the article © 2016 Maia et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Maia et al. Parasites & Vectors (2016) 9:170 Page 2 of 6 the genus Phlebotomus and of the last two by mosquitoes shelter) were studied (Table 1). The dogs were from the mainly from the genera Culex, Aedes and Anopheles [2, 3]. districts of Setúbal (n = 68, including 13 dogs from the Other less known filarial worms endemic in Europe such contiguous districts of Évora and Beja) and Faro (n = as Acanthocheilonema dracunculoides and Acanthocheilo- 162). Domestic dogs were included after informed con- nema reconditum are transmitted mainly by ticks or by sent was obtained from the owners. In the case of stray fleas and lice, respectively [4]. dogs, a written consent for enrolment was obtained from Endosymbiont bacteria Wolbachia spp. (order Rickett- their legal detainer, i.e. the person in charge of the res- siales) have been found in several filarial species, such as cue association. D. immitis and D. repens, but not in Achanthocheilonema Whenever available, data on gender, breed, age, life style, spp. [5]. Wolbachia organisms seem to play an essential living conditions, prophylactic use of sand fly and/or mos- role in the biology of filarial parasites and in the pathogen- quito repellents and of macrocyclic lactones, and clinical esis of infections due to these nematodes, potentially in- status, i.e. absence (Table 1) or presence (Table 2) of at creasing the severity of clinical signs [6, 7]. least one sign compatible with leishmaniosis or dirofilario- Canine leishmaniosis is a systemic chronic condition sis) were registered for each dog. Clinical signs comprised whose clinical manifestations often include lymphadeno- anorexia, cough, dermatological manifestations, dyspnoea, megaly, cutaneous alterations, weight loss, ocular signs, epistaxis, exercise intolerance, gastrointestinal alterations, epistaxis, onychogryphosis and lameness [8]. Canine diro- lameness, lethargy, lymphadenopathy, muscular atrophy, filariosis is associated with a dry chronic cough, exercise onychogryphosis, ocular manifestations, pale mucous and intolerance, dyspnoea, weakness, weight loss, epistaxis, weight loss. cyanosis and even congestive heart failure [7]. Both para- This study was ethically approved by the boards of the sitoses are endemic in the south of Portugal. Leishmania IHMT-UNL and of the Faculty of Veterinary Medicine seroprevalence in dogs has ranged from 3.8 % in randomly (ULHT) as complying with the Portuguese legislation for screened apparently healthy animals from the Algarve [9] the protection of animals (Decree-Law no. 113/2013). to 40.6 % in dogs from the same region that were clinically Whole blood samples were collected by cephalic or suspect of leishmaniosis [10]. The detection of D. immitis jugular venipuncture and spotted onto filter paper. Sam- antigen has ranged from 2.4 % in apparently healthy dogs ples were dried at room temperature and kept at 4 °C from Lisbon to 17.1 % in dogs from the Algarve with clin- until DNA extraction by a commercial kit (Kit Citogene®, ical signs compatible with a CVBD [9]. Wolbachia Citomed). Four discs of filter paper (4 mm in diameter spp. DNA has also been detected in D. immitis infected each) were incubated with lysis buffer (150 μl) and 1.5 μl dogs from the centre and south of Portugal [11, 12], and of proteinase K (20 mg/ml). Further DNA extraction A. reconditum and A. drancunculoides infections have followed the kit manufacturer’s instructions. been reported in animals from the same areas [13, 14]. All the samples were submitted for real-time PCR (qPCR) Furthermore, D. repens microfilariae were recently de- for L. infantum, filariae (D. immitis, D. repens, A. dracuncu- tected in one dog from the Algarve region, the southern- loides and A. reconditum) and Wolbachia spp. Leishmania most region of continental Portugal [15]. quantitative PCR was performed as previously described by Vector-borne pathogen co-infections may lead to an Francino et al. [17]. qPCRs for Spirurida (Filariidae, Oncho- increased severity of clinical signs as previously shown cercidade and Thelaziidae) and Wolbachia were carried out in dogs from southeastern Spain with leishmaniosis and/ in a final volume of 20 μL using SYBR Select (Life Tech- or filariosis [16]. On the other hand, a protective role of nologies), 0.1 μM of each primer for Wolbachia species (de- Wolbachia limiting the severity of leishmaniosis was also scribed by Tabar et al. [16]) and 0.5 μM of each primer for observed in dogs co-infected with L. infantum and D. Spirurida (5′-CGTAATTTTARTWCTTCTTTTTATGAT immitis [16]. As the presence of co-infections may lead RCTA-3′; 5′-CCAAAYAAACGWTCCTTATCAGTYAA- to a non-characteristic clinical outcome which will fur- 3′) and 4 μL of DNA (10–100 ng). The thermal cycling ther complicate the diagnosis, treatment and prognosis, profile was 50 °C for 2 min and 95 °C for 10 min followed together with the zoonotic potential of L. infantum and by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The Dirofilaria spp., the aim of this work was to evaluate the eukaryotic 18S RNA Pre-Developed TaqMan Assay Re- prevalence of mono- and co-infections by L. infantum, agent (Life Technologies) was used as internal reference for filariae and Wolbachia and their association with clinical dog genomic DNA amplification to ensure proper qPCR signs consistent with leishmaniosis or dirofilariosis in amplification of each sample and that negative results cor- dogs from the south of Portugal. responded to true negative samples rather than to a prob- From May 2011 to February 2014, a total of 230 dogs lem with DNA loading, sample degradation or PCR from veterinary medical centres and animal shelters in inhibition. Positive qPCR controls were obtained from clin- southern Portugal randomly selected (i.e. out of any dog ical samples that had been previously amplified and se- present to the veterinary clinic or any dog living in the quenced to confirm the pathogen and water was used as a Maia et al. Parasites & Vectors (2016) 9:170 Page 3 of 6 Table 1 Molecular prevalence of L. infantum, filariae and Wolbachia among dogs with no clinical signs compatible with leishmaniosis or dirofilariosis (n = 155) Variable/category No. (%) of Percentage (n) of positive dogs tested dogs PCR PCR PCR ≥1 agent L. infantum filariae Wolbachia spp. Gender 154 Female 84 (54.5) 70.2 (59) 13.1 (11) 6.0 (5) 72.6 (61) Male 70 (45.5) 68.6 (48) 11.4 (8) 7.1 (5) 70.0 (49) Age (months) 143 [1–11] 23 (16.1) 60.9 (14) 4.3 (1) 0.0 (0) 60.9 (14) [12–83] 82 (57.3) 74.4 (61) 13.4 (11) 6.1 (5) 76.8 (63) [84–192] 38 (26.6) 60.5 (23) 15.8 (6) 13.2 (5) 63.2 (24) Breed 147 Defined (incl. crosses) 68 (46.3) 58.8 (40)a 11.8 (8) 5.9 (4) 63.2 (43) a Mongrel 79 (53.7) 77.2 (61) 12.7 (10) 7.6 (6) 77.2 (61) Aptitude 155 Pet 108 (69.7) 76.9 (83)b 12.0 (13) 5.6 (6) 79.6 (86)c 1 b Other 47(30.3) 51.1 (24) 12.8 (6) 8.5 (4) 51.1 (24)c Housing 145 Indoors and mixed 75 (51.7) 61.3 (46) 9.3 (7) 5.3 (4) 64.0 (48) Outdoors 70 (48.3) 75.7 (53) 17.1 (12) 8.6 (6) 77.1 (54) Repellent insecticides2 87 Yes 30 (34.5) 54.4 (31) 8.8 (5) 3.5 (2) 57.9 (33) No 57 (65.5) 43.3 (13) 10.0 (3) 6.7 (2) 46.7 (14) Macrocyclic lactones3 54 Yes 13 (24.1) 38.5 (5) 0.0 (0) 0.0 (0) 38.5 (5) No 41 (75.9) 31.7 (13) 17.1 (7) 14.6 (6) 36.6 (15) Total 155 (100) 69.0 (107) 12.3 (19*) 6.5 (10) 71.0 (110) 1 Farm, guard, hunting, shepherd or stray; 2Deltamethrin, flumethrin and/or permethrin; 3Ivermectin, mimemycin oxime or moxydectin; aP = 0.026; bP = 0.003; cP = 0.001; *Comprising 18 cases of D. immitis infection and one case of A. dracunculoides and D. immitis co-infection PCR-negative control. The L. infantum load in blood was filarial infections in southern Portugal had either been quantified according to Delta Ct, namely: low DeltaCt > 15; carried out exclusively with shelter dogs [13, 14] or with medium DeltaCt 5-15; high DeltaCt < 5 and very high Del- not so many (n = 157) domestic dogs [12]. taCt negative. Spirurida qPCR positive samples were se- One hundred and thirty-nine dogs were qPCR-positive quenced by the Big-Dye Terminator Cycle Sequencing to L. infantum. Out of the 97 dogs tested from November Ready reaction Kit (Life Technologies) using the same to April (Leishmania non-transmission period) 72 (74.2 %; primers. Sequences obtained were compared with GenBank CI: 64.3–82.6 %) were positive, whereas from May to database (www.ncbi.nlm.nih.gov/BLAST). October (transmission period) L. infantum DNA was de- Percentages of positivity for L. infantum, filariae and tected in 67 (50.4 %; CI: 41.6–59.2 %) of 133 canine blood Wolbachia spp. regarding the independent variables and samples (P < 0.001). The significantly higher (P < 0.001) categories were compared by the Chi-square or Fisher’s Leishmania prevalence obtained in the present study, i.e. exact tests. A P value ≤ 0.05 was considered as statisti- 69.0 % (CI: 61.1–76.2 %) in dogs with no clinical signs of cally significant. Exact binomial 95 % confidence inter- leishmaniosis and/or dirofilariosis (Table 1) and 42.7 % vals (CI) were defined for the proportions. Analyses (CI: 31.3–54.6 %) in dogs with compatible clinical signs were performed with SPSS® 21 software for Windows (Table 2) compared with the 1.1 % recently obtained in and with StatLib. dogs from the south of the country by conventional PCR This study was carried out with a considerable number [18], could be related to the higher sensitivity of the qPCR, of dogs (n = 230), which were mainly owned and tame which is able to detect less than one parasite per millilitre ones. On the contrary, previous investigations on canine of blood [17]. This most recent figure of 69.0 % is more in Maia et al. Parasites & Vectors (2016) 9:170 Page 4 of 6 Table 2 Molecular prevalence of L. infantum, filariae and Wolbachia in dogs with clinical signs compatible with leishmaniosis and/or dirofilariosis (n = 75) Variable/category No. (%) of Percentage (n) of positive dogs tested dogs PCR PCR PCR ≥1 agent L. infantum filariae Wolbachia spp. Gender 75 Female 32 (42.7) 28.1 (9)a 12.5 (4) 9.4 (3) 34.4 (11) Male 43 (57.3) 53.5 (23)a 7.0 (3) 7.0 (3) 55.8 (24) Age (months) 67 [1–11] 1 (1.5) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) [12–83] 33 (49.3) 39.4 (13) 12.1 (4) 12.1 (4) 45.5 (15) [84–192] 33 (49.3) 42.4 (14) 9.1 (3) 6.1 (2) 45.5 (15) Breed 73 Defined (incl. crosses) 40 (54.8) 40.0 (16) 7.5 (3) 7.5 (3) 42.5 (17) Mongrel 33 (45.2) 48.5 (16) 12.1 (4) 9.1 (3) 54.5 (18) Aptitude 75 Pet 37 (49.3) 40.5 (15) 16.2 (6) 13.5 (5) 48.6 (18) Other1 38 (50.7) 44.7 (17) 2.6 (1) 2.6 (1) 44.7 (17) Housing 41 Indoors and mixed 18 (43.9) 27.8 (5) 5.6 (1) 5.6 (1) 33.3 (6) Outdoors 23 (56.1) 47.8 (11) 17.4 (4) 13.0 (3) 52.2 (12) Repellent insecticides2 55 Yes 19 (34.5) 21.1 (4)b 10.5 (2) 5.3 (1) 26.3 (5) No 36 (65.5) 52.8 (19)b 5.6 (2) 5.6 (2) 55.6 (20) Macrocyclic lactones3 37 Yes 9 (24.3) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) No 28 (75.7) 25.0 (7) 10.7 (3) 7.1 (2) 32.1 (9) Total 75 (100) 42.7 (32) 9.3 (7*) 8.0 (6) 46.7 (35) 1 Farm, guard, hunting or stray; 2Deltamethrin or permethrin; 3Ivermectin, milbemycin oxime or moxydectin; aP = 0.050; bP = 0.048; *Comprising six dogs positive for D. immitis and another one for A. reconditum accordance with previously reported molecular prevalence clinical signs was significantly higher in pets in compari- values clearly above 50 % [16]. Nevertheless, it cannot be son with dogs with other aptitudes (Table 1). The ruled out that positive blood results were due to transient present results, therefore, disagree with those that re- contamination during the transmission season and may vealed a higher prevalence of infection in guard dogs as thus represent just exposure to the parasite rather than an compared to pets in the Madrid region, Spain [19]. established infection. These latter findings might be somewhat due to a differ- A negative association was found between Leishmania ent lifestyle of pets (i.e. urban vs. rural) in distinct geo- positivity and clinical signs compatible with leishmanio- graphic areas. The higher prevalence of L. infantum sis and/or dirofilariosis (69.0 % [Table 1] versus 42.7 % DNA in clinically healthy mongrel dogs in comparison [Table 2]; P < 0.001). The fact that more dogs considered with those belonging to defined breeds or their crosses clinically healthy harboured Leishmania parasites in (P = 0.026) might be related with a certain level of resist- their circulation might be due to a fact most of the en- ance to disease development and progression by the rolled dogs were randomly selected from those attending mixed-breed infected dogs [20]. veterinarian clinics and, therefore, probably receiving In those dogs showing clinical signs compatible with more medical care, better nutrition, and for that reason leishmaniosis and/or dirofilariosis the detection of L. were less prone to develop disease. The prevalence of L. infantum DNA in the blood was significantly higher (P infantum (76.9 %; CI: 67.7–84.4 %; P = 0.003) and that of = 0.050; Table 2) in males (53.5 %; CI: 37.6–68.8 %). infection with at least one pathogen (≥1 agent; 79.6 %; Gender predisposition to the infection has been consid- CI: 70.8–86.8 %; P = 0.001) in dogs without compatible ered by some authors as a non-determinant factor for Maia et al. Parasites & Vectors (2016) 9:170 Page 5 of 6 leishmaniosis [19, 20], while others have reported a in endemic areas a combined PCR for Wolbachia and higher prevalence in male dogs [21, 22]. On the other Dirofilaria should be performed for the diagnosis of hand, the use of long-acting topical insecticides on dogs heartworm, in order to avoid a high percentage of false- has been shown to be an effective measure in reducing negative Dirofilaria results due to the lack of testing for the prevalence of Leishmania infection [23]. Therefore, the endosymbiont. it is not entirely surprising that the prevalence of Leish- Wolbachia DNA was detected in 5 (31.3 %; CI: 11.0– mania was higher in those dogs not protected against 58.7 %) dogs solely infected with D. immitis and in 11 insects (P = 0.048; Table 2). In fact, and although the (68.8 %; CI: 41.3–89.0 %; P = 0.034) co-infected dogs (11 number of effective molecules for prophylaxis against with L. infantum-D. immitis and one with A. leishmaniosis has increased in the last few years, our re- dracunculoides-D.immitis), reinforcing that this endo- sults clearly reinforce the idea that there is still a long symbiont does not seem to be present in Achanthochei- way to go regarding the prevention and control of Leish- lonema spp. alone [5]. On the other hand, our results mania infection. contrast with a previous study carried out in dogs with Filarial infections were detected in 26 (11.3 %; CI: 7.5– leishmaniosis and/or dirofilariosis from Spain where the 16.1 %) blood samples: 24 (10.4 %; CI: 6.8–15.1 %) dogs prevalence of the endosymbiont was significantly lower harboured D. immitis only, one (0.4 %; CI: 0.01–2.4 %) in microfilariaemic dogs co-infected with L. infantum dog A. reconditum only and another one (0.4 %; CI: 0.01– [16]. Interestingly, in the present study only three out of 2.4 %) A. drancunculoides in co-infection with D. immitis. the 11 Wolbachia, D. immitis and L. infantum co- This corroborates the previous reports on the circulation infected dogs presented clinical signs compatible with of these nematodes in the south and central regions of leishmaniosis and/or dirofilariosis, which might have Portugal [9, 12–14]. D. immitis prevalence in the present been due to the stimulation of a Th1 type protective- study (10.9 %; CI: 7.2–15.6 %) was lower (although non- immune response triggered by the nematode and the significantly; P = 0.111) than the overall prevalence of endosymbiont bacterium [16, 24]. 15.1 % (105/696) recently obtained in three coastal areas In the present study, 19 (8.3 %; CI: 5.0–12.6 %) dogs of central and southern-central Portugal [14]. Differences were co-infected with L. infantum and D. immitis. It can be related to the dynamics of infection over time, should be stressed that the occurrence of co-infections which is probably linked to the distribution and abun- with these vector-borne agents, which are relatively dance of the vectors and to the surveyed canine popula- common in dogs living in geographic areas where com- tion. In fact, in the work performed by Alho et al. [14] all petent vectors for the different pathogens co-exist, might the screened animals were sheltered dogs that lived out- induce more severe and atypical clinical outcomes that doors and had probably not received any prophylactic will further complicate diagnosis, treatment and progno- measure for heartworm infection, thus being more ex- sis [9, 16, 25]. However, this hypothesis has not been posed to vectors and to the agents they might transmit. corroborated in the present study and the reason might On the other hand, in the present study most of the tested be the random screening of the canine population, with dogs were pets living partially indoors and receiving veter- most dogs showing no clinical signs, even when they inary health care. Interestingly, and although the differ- were positive for one or more of the studied agents. ence was not statistically significant (P = 0.562), D. immitis The on-going and recurrent detection of zoonotic DNA was not detected in any of the dogs receiving vector-borne agents in dogs with or without clinical macrocyclic lactones, suggesting the importance of using signs reinforces the importance of increasing the veter- this prophylactic measure in areas where dirofilariosis is inary community, owners and public health authoritiesʼ endemic. As the compliance to the use of ectoparasiticides awareness regarding the risk of infection. It also high- was not evaluated, it cannot be ruled out whether the rea- lights the need to apply efficient prophylactic measures, son for no differences between the use of insecticides/re- such as insect repellents and macrocyclic lactones (in- pellents and the presence of heartworm infection was due cluding compliance to administration), not only by the to irregular administration of insecticides. local owners but also by the tourists from non-endemic The detection of Wolbachia DNA in 16 (64.0 %; CI: countries vacationing with their dogs in endemic areas, 42.5–82.0 %) dogs positive to D. immitis was higher than in order to prevent infection and disease among mam- the 52.6 % (20/38; P = 0.372) and 30.6 % (15/49; P = malian hosts including humans. Nevertheless, as envir- 0.006) recently obtained in Portuguese [11] and Spanish onmental changes, global warming, and growing world [16] dogs, respectively. As reported by previous publica- trade and animal transportation, including the increased tions, only D. immitis and D. repens have ever been mobility of dogs across borders, have an impact on the found infected with Wolbachia, but not all the speci- geographic distribution, abundance and vectorial cap- mens of these filarial parasites transport the endosymbi- acity of arthropods, the expansion of L. infantum and fil- ont bacteria. Taken together, these findings suggest that ariae to new locations should not be neglected. The Maia et al. Parasites & Vectors (2016) 9:170 Page 6 of 6 present results further stress the need for sustained de- and canine leishmaniasis in an endemic European area, the Algarve Region velopment of multi-pathogen detection methods in re- in Portugal. Mem Inst Oswaldo Cruz. 2013;108:481–7. 11. Landum M, Ferreira CC, Calado M, Alho AM, Maurício IL, Meireles JS, et al. gions endemic and even non-endemic for CVBD. Detection of Wolbachia in Dirofilaria infected dogs in Portugal. Vet Parasitol. 2014;204:407–10. Competing interests 12. Maia C, Coimbra M, Ramos C, Cristóvão JM, Cardoso L, Campino L. The authors declare that they have no competing interests. Serological investigation of Leishmania infantum. Dirofilaria immitis and Angiostrongylus vasorum in dogs from southern Portugal. Parasit Authors’ contributions Vectors. 2015;8:152. CM planned, designed and supervised the study, and wrote the manuscript; 13. Menn B, Lorentz S, Naucke TJ. Imported and travelling dogs as carriers of LA and LS performed qPCR assays; JMC performed DNA extraction; LuC canine vector-borne pathogens in Germany. Parasit Vectors. 2010;3:34. performed data analysis and revised the manuscript; MDT, OF and LeC 14. Alho AM, Landum M, Ferreira C, Meireles J, Gonçalves L, de Carvalho LM, et reviewed the manuscript; XR planned the study and reviewed the al. Prevalence and seasonal variations of canine dirofilariosis in Portugal. Vet manuscript. All authors read and approved the final manuscript. Parasitol. 2014;206:99–105. 15. Maia C, Lorentz S, Cardoso L, Otranto D, Naucke TJ. Detection of Dirofilaria Acknowledgements repens microfilariae in a dog from Portugal. Parasitol Res. 2015;115:441–3. This work was partially supported by Funds to GHTM (UID/Multi/04413/ 16. Tabar MD, Altet L, Martínez V, Roura X. Wolbachia, filariae and 2013). The authors thank the cooperation of veterinarians, auxiliary staff, dog Leishmania coinfection in dogs from a Mediterranean area. J Small owners and shelters that contributed with collection of samples. The work of Anim Pract. 2013;54:174–8. C. Maia and L. Cardoso was done under the frame of EurNegVec COST 17. Francino O, Altet L, Sánchez-Robert E, Rodriguez A, Solano-Gallego L, Action TD1303. Alberola J, et al. Advantages of real-time PCR assay for diagnosis and Publication of this paper has been sponsored by Bayer Animal Health in the monitoring of canine leishmaniosis. Vet Parasitol. 2006;137:214–21. framework of the 11th CVBD World Forum Symposium. 18. Maia C, Almeida B, Coimbra M, Fernandes MC, Cristóvão JM, Ramos C, et al. Bacterial and protozoal agents of canine vector-borne diseases in the blood of Author details domestic and stray dogs from southern Portugal. Parasit Vectors. 2015;8:138. 1 Global Health and Tropical Medicine, Medical Parasitology Unit, Instituto de 19. Galvez R, Miró G, Descalzo MA, Nieto J, Dado D, Martín O, et al. Emerging Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal. trends in the seroprevalence of canine leishmaniasis in the Madrid region 2 Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e (central Spain). Vet Parasitol. 2010;169:327–34. Tecnologias, Lisbon, Portugal. 3Vetgenomics, Parc de Recerca Universitat 20. 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Vet Parasitol. 2001;98:215–38. 6. Kramer L, Grandi G, Leoni M, Passeri B, McCall J, Genchi C, et al. Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis Submit your next manuscript to BioMed Central infection. Vet Parasitol. 2008;158:191–5. and we will help you at every step: 7. McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. Adv Parasitol. 2008;66:193–285. • We accept pre-submission inquiries 8. Campino L, Maia C. The role of reservoirs: canine leishmaniasis. In: Ponte- • Our selector tool helps you to find the most relevant journal Sucre A, Padron-Nieves M, Diaz E, editors. Drug resistance in Leishmania • We provide round the clock customer support parasites – consequences, molecular mechanism and possible treatments. Vienna: Springer Verlag; 2013. p. 45–64. • Convenient online submission 9. Cardoso L, Mendão C, Madeira de Carvalho L. Prevalence of Dirofilaria • Thorough peer review immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and • Inclusion in PubMed and all major indexing services Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal – a national serological study. Parasit Vectors. 2012;5:62. • Maximum visibility for your research 10. Maia C, Dionísio L, Afonso MO, Neto L, Cristóvão JM, Campino L. Leishmania infection and host-blood feeding preferences of phlebotomine sandflies Submit your manuscript at www.biomedcentral.com/submit