Serological investigation of Leishmania infantum, Dirofilaria immitis and Angiostrongylus vasorum in dogs from southern Portugal
© Maia et al.; licensee BioMed Central. 2015
Received: 3 December 2014
Accepted: 25 February 2015
Published: 23 March 2015
Leishmaniosis, dirofilariosis and angiostrongylosis are parasitic diseases of established importance in dogs worldwide. The aim of the present report was to determine all together levels of infection with or exposure to Leishmania infantum, Dirofilaria immitis and Angiostrongylus vasorum in dogs from the Algarve region, in southern Portugal.
Serum samples were obtained from a total of 170 apparently healthy dogs. Antibodies to L. infantum were detected by the direct agglutination test; and antigens of D. immitis and of A. vasorum by an enzyme-linked immunosorbent assay and an immunochromatography test, respectively. Antibodies to L. infantum were found in 18.2% (31/170) of dogs, while D. immitis antigen was detected in 9.4% (16/170). None of 120 dogs tested out of the 170 under investigation had evidence of infection with A. vasorum. D. immitis infection was more prevalent in dogs aged 84–204 months than in those younger than 12 months. Three dogs were positive to both L. infantum and D. immitis.
Dogs living in southern Portugal are at risk of leishmaniosis and dirofilariosis. This scenario should make the veterinary community, local dog owners and also tourists from non-endemic countries coming on vacation with their pets aware of the need of effective prophylactic measures, in order to protect animals and public health.
KeywordsLeishmania infantum Dirofilaria immitis Angiostrongylus vasorum Dog Portugal Seroprevalence
Leishmaniosis, dirofilariosis and angiostrongylosis are parasitic diseases of established importance in dogs worldwide. Leishmaniosis caused by the protozoan Leishmania infantum (Trypanosomatida, Trypanosomatidae) and heartworm disease caused by the nematode Dirofilaria immitis (Spirurida, Onchocercidae) are vector-borne zoonoses with importance in veterinary medicine and widely present in the Mediterranean [1-3]. Both parasitoses are endemic in Portugal, with an overall national seroprevalence of 6.3%  and 3.6-8.9% , respectively.
Canine leishmaniosis is a systemic chronic condition whose clinical manifestations usually include lymphadenomegaly, cutaneous alterations, loss of body weight, ocular signs, epistaxis, onychogryphosis and lameness . Canine cardiopulmonary dirofilariosis is associated with a dry chronic cough, exercise intolerance, dyspnoea, weakness, weight loss, epistaxis, cyanosis and even congestive heart failure .
Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum (Strongylida, Angiostrongylidae), is an emerging disease with an increasing number of cases diagnosed in the last few years, and with a high established relevance in several regions of Europe . The disease is associated with an array of clinical problems, including cardiorespiratory, coagulopathic and neurologic signs . Albeit the laboratory Baermann method, using faeces, is the gold-standard to detect first-stage larvae (L1), serological techniques for the detection of circulating A. vasorum antigens or specific antibodies have recently been developed [9,10]. In Europe prevalence as assessed by the detection of circulating parasite antigens in dogs has been found to range from 0.5% in Germany to 1.3% in the United Kingdom, while the detection of specific antibodies ranged from 1.3% in Poland to 3.2% in the UK [11,12]. In coastal areas of south-central Portugal, 1.8% (6/341) of shelter dogs were antigen-positive, while antibodies against the parasite were detected in 2.4% of those dogs .
The present report aimed at determining the levels of infection with or exposure to vector-borne parasites (L. infantum and D. immitis) and parasites acquired by ingestion of intermediate hosts (A. vasorum) in dogs from the Algarve region, the southernmost part of continental Portugal, in view of the scarcity of epidemiological studies targeting these parasites all together in canine populations.
Prevalence of antibodies to Leishmania infantum in dogs from southern Portugal according to independent variables and their categories
No. (%) of dogs tested
% of positive
p = 0.161
p = 0.747
p = 0.393
Medium or long
p = 0.530
p = 1.0
p = 0.280
Exclusively or mainly indoors
Mainly or exclusively outdoors
Sand fly repellents*
p = 1.0
p = 1.0
Prevalence of Dirofilaria immitis antigen in dogs from southern Portugal according to independent variables and their categories
No. (%) of dogs tested
% of positive
p = 0.297
p = 0.071
p = 1.0
Medium or long
p = ND
p = 0.352
p = 1.0
Exclusively or mainly indoors
Mainly or exclusively outdoors
p = 0.128
p = 1.0
This study was approved by the ethics board of the Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa (IHMT-UNL) as complying with the Portuguese legislation for the protection of animals (Law no. 92/1995).
The direct agglutination test (DAT) for titration of specific antibodies was performed in 170 dogs using a standard freeze-dried Leishmania antigen as previously described . The DAT cut-off was established at a titre of 400 . D. immitis antigen was detected in 170 animals by using a commercial enzyme-linked immunosorbent assay kit (PetChek Canine Heartworm Antigen Test®, IDEXX Laboratories, Westbrook, ME, USA). Detection of A. vasorum antigens was performed in only 120 dogs out of the 170 under investigation by using a commercial immunochromatography test (Angio Detect™ Test, IDEXX Laboratories).
The exact binomial test established confidence intervals (CI) with a 95% confidence level. The chi-square or Fisher’s exact tests were used to compare percentages of positivity among categories of the same independent variables and also the total prevalence of each agent . A p value < 0.05 was considered as statistically significant. Analyses were performed with StatLib and SPSS® 21 software for Windows.
Antibodies to L. infantum were found in 31 (18.2%) dogs, at the dilutions of 1:400 (n = 5), 1:800 (n = 2), 1:1,600 (n = 2), 1:3,200 (n = 5), 1:6,400 (n = 6), 1:12,800 (n = 6), 1:25,600 (n = 4) and 1:51,200 (n = 1) (Table 1). The seroprevalence of Leishmania found in the present study was higher than the ones obtained in randomly screened animals (4.7%)  and in clinically healthy dogs (3.8%)  from the Algarve region that were presented to veterinary clinics, suggesting an apparent increase in the level of Leishmania infection in the Algarve .
D. immitis antigen was detected in 16 (9.6%) animals (Table 2). This prevalence is apparently higher than the values recently obtained in three coastal areas of south-central Portugal (7.9%)  and in other areas further north (2.1%) , suggesting that the climatic conditions in the southern regions of the country might be more favourable to the proliferation and abundance of vectors of D. immitis [19,20]. An exception to this trend was reported in Figueira da Foz, located in the coastal edge of the central region of Portugal, where 25.7% of the dogs were found infected .
Prevalence of D. immitis infection was significantly higher in dogs aged 84–204 months than in dogs younger than 12 months (p = 0.021), which may be explained by a cumulative exposure of older animals to the parasite through an increased contact with the vectors [5,21]. Although the difference was not statistically significant (p = 0.128), D. immitis antigens were not detected in any of the animals receiving macrocyclic lactones; anyhow, this finding suggests the importance of using prophylaxis in areas where dirofilariosis is endemic.
Cross-reactivity between D. immitis and A. vasorum in dogs experimentally infected with the latter has previously been reported for some heartworm antigen test kits . However, in the present study no cross-reaction between both nematodes seem to have happened, as A. vasorum antigens were not detected in any of the dogs tested (0.0%; 95% CI: 0.0-3.0). The non-detection of this parasite, in comparison with the survey (1.8%) carried out in other coastal areas of Portugal , might be related with the sensitivity of the present test (i.e. 98.1% ), with seasonal factors and/or with the surveyed canine populations, as in the previous work all animals were shelter dogs, probably at a higher risk of contact with the intermediate host (i.e. snails and slugs), while in the present study most of the dogs were pets. Nevertheless, larger surveys and using different techniques (Baerman method, detection of specific antibodies and antigens) are necessary to gain more information on the diffusion of A. vasorum, for which the actual national distribution is poorly acquainted.
In the present study, no statistical association was found between positivities to L. infantum and D. immitis. Only three dogs were positive to both L. infantum and D. immitis. Co-infections with these two vector-borne pathogens are relatively frequent in dogs living in geographic areas where the presence of competent vectors for the different pathogens overlap [5,24]. Although in this study the three animals positive to both parasites were apparently healthy, it is important to keep in mind that the occurrence of co-infections with vector-borne pathogens might induce uncharacteristic clinical outcomes that will further complicate diagnosis, treatment and prognosis .
The findings of this study reveal that dogs from southern Portugal are at risk of acquiring two of the three parasites assessed (i.e. the agents of leishmaniosis and dirofilariosis). They also reinforce the importance to alert the veterinary community, local dog owners as well as tourists from non-endemic countries coming on vacation with their pets to the need of prophylactic measures, such as insect repellents and macrocyclic lactones, in order to defend animals and public health.
This work was supported by Centro de Malária e outras Doenças Tropicais, IHMT-UNL, Portugal, and EU grant FP7-261504 EDENext, and is catalogued by the EDENext Steering Committee as EDENext 294 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The authors thank the cooperation of veterinarians, auxiliary staff, dog owners and shelters that contributed with or allowed collection of samples, and also acknowledge Servive Portugal (Valença, Portugal) for their assistance with the acquisition of Angio Detect™ Test. C. Maia (SFRH/BPD/44082/2008) holds a scholarship from Fundação para a Ciência e a Tecnologia, Ministério da Educação e Ciência, Portugal. The work of C. Maia and L. Cardoso was done under the frame of EurNegVec COST Action TD1303.
Publication of this paper has been sponsored by Bayer HealthCare – Animal Health Division in the framework of the 10th CVBD World Forum Symposium.
- Capelli G, Frangipane di Regalbono A, Simonato G, Cassini R, Cazzin S, Cancrini G, et al. Risk of canine and human exposure to Dirofilaria immitis infected mosquitoes in endemic areas of Italy. Parasit Vectors. 2013;6:60.View ArticlePubMed CentralPubMedGoogle Scholar
- Miró G, Montoya A, Roura X, Gálvez R, Sainz A. Seropositivity rates for agents of canine vector-borne diseases in Spain: a multicentre study. Parasit Vectors. 2013;6:117.View ArticlePubMed CentralPubMedGoogle Scholar
- Genchi C, Bowman D, Drake J. Canine heartworm disease (Dirofilaria immitis) in Western Europe: survey of veterinary awareness and perceptions. Parasit Vectors. 2014;7:206.View ArticlePubMed CentralPubMedGoogle Scholar
- Cortes S, Vaz Y, Neves R, Maia C, Cardoso L, Campino L. Risk factors for canine leishmaniasis in an endemic Mediterranean region. Vet Parasitol. 2012;189:189–96.View ArticlePubMedGoogle Scholar
- Cardoso L, Mendão C, Carvalho LM. Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal – a national serological study. Parasit Vectors. 2012;5:62.View ArticlePubMed CentralPubMedGoogle Scholar
- Campino L, Maia C. The role of reservoirs: canine leishmaniasis. In: Ponte-Sucre A, Padron-Nieves M, Diaz E, editors. Drug resistance in Leishmania parasites – consequences, molecular mechanism and possible treatments. Vienna: Springer Verlag; 2012. p. 45–64.Google Scholar
- McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. Adv Parasitol. 2008;66:193–285.View ArticlePubMedGoogle Scholar
- Helm JR, Morgan ER, Jackson MW, Wotton P, Bell R. Canine angiostrongylosis: an emerging disease in Europe. J Vet Emerg Crit Care. 2010;20:98–109.View ArticleGoogle Scholar
- Schnyder M, Tanner I, Webster P, Barutzki D, Deplazes P. An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs. Vet Parasitol. 2011;179:152–8.View ArticlePubMedGoogle Scholar
- Schnyder M, Stebler K, Naucke TJ, Lorentz S, Deplazes P. Evaluation of a rapid device for serological in-clinic diagnosis of canine angiostrongylosis. Parasit Vectors. 2014;7:72.View ArticlePubMed CentralPubMedGoogle Scholar
- Schnyder M, Schaper R, Bilbrough G, Morgan ER, Deplazes P. Seroepidemiological survey for canine angiostrongylosis in dogs from Germany and the UK using combined detection of Angiostrongylus vasorum antigen and specific antibodies. Parasitology. 2013;140:1442–50.View ArticlePubMed CentralPubMedGoogle Scholar
- Schnyder M, Schaper R, Pantchev N, Kowalska D, Szwedko A, Deplazes P. Serological detection of circulating Angiostrongylus vasorum antigen- and parasite-specific antibodies in dogs from Poland. Parasitol Res. 2013;112:109–17.View ArticlePubMedGoogle Scholar
- Alho AM, Schnyder M, Meireles J, Belo S, Deplazes P, Carvalho LM. Preliminary results on the seroprevalence of Angiostrongylus vasorum and co-infection with Dirofilaria immitis in shelter dogs from Portugal [abstract]. Parasit Vectors. 2014;7 Suppl 1:O26.View ArticlePubMed CentralGoogle Scholar
- Schallig HD, Cardoso L, Semião-Santos SJ. Seroepidemiology of canine leishmaniosis in Évora (southern Portugal): 20-year trends. Parasit Vectors. 2013;6:100.View ArticlePubMed CentralPubMedGoogle Scholar
- Maia C, Nunes M, Cristóvão J, Campino L. Experimental canine leishmaniasis: clinical, parasitological and serological follow-up. Acta Trop. 2010;116:193–9.View ArticlePubMedGoogle Scholar
- Petrie A, Watson P. Statistics for veterinary and animal science. 2nd ed. Oxford: Blackwell Publishing; 2006.Google Scholar
- Maia C, Dionísio L, Afonso MO, Neto L, Cristóvão JM, Campino L. Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal. Mem Inst Oswaldo Cruz. 2013;108:481–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Vieira L, Silvestre-Ferreira AC, Fontes-Sousa AP, Balreira AC, Morchón R, Carretón E, et al. Seroprevalence of heartworm (Dirofilaria immitis) in feline and canine hosts from central and northern Portugal. J Helminthol. 2014;14:1–5.View ArticleGoogle Scholar
- Alho AM, Nunes T, Rinaldi L, Meireles J, Belo S, Deplazes P, et al. Transmission risk of dirofilariosis in Portugal [abstract]. Parasit Vectors. 2014;7(Suppl1):O16.View ArticlePubMed CentralGoogle Scholar
- Roiz D, Ruiz S, Soriguer R, Figuerola J. Climatic effects on mosquito abundance in Mediterranean wetlands. Parasit Vectors. 2014;7:333.View ArticlePubMed CentralPubMedGoogle Scholar
- Vieira AL, Vieira MJ, Oliveira JM, Simões AR, Diez-Baños P, Gestal J. Prevalence of canine heartworm (Dirofilaria immitis) disease in dogs of central Portugal. Parasite. 2014;21:5.View ArticlePubMed CentralPubMedGoogle Scholar
- Schnyder M, Deplazes P. Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits. Parasit Vectors. 2012;5:258.View ArticlePubMed CentralPubMedGoogle Scholar
- IDEXX. Angio Detect™ Test – the only in-clinic antigen detection test for Angiostrongylus vasorum. Westbrook: IDEXX Laboratories, 2015.Google Scholar
- Tabar MD, Altet L, Martínez V, Roura X. Wolbachia, filariae and Leishmania coinfection in dogs from a Mediterranean area. J Small Anim Pract. 2013;54:174–8.View ArticlePubMedGoogle Scholar
- De Tommasi AS, Otranto D, Dantas-Torres F, Capelli G, Breitschwerdt EB, de Caprariis D. Are vector-borne pathogen co-infections complicating the clinical presentation in dogs? Parasit Vectors. 2013;6:97.View ArticlePubMed CentralPubMedGoogle Scholar
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