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The occurrence of the filarial nematode Dirofilaria repens in canine hosts from Maio Island, Cape Verde

Published online by Cambridge University Press:  01 February 2016

R. Marcos,
C. Pereira,
J.P. Maia,
M. Santos,
C. Luzzago,
S. Lauzi,
C. Genchi,
A. Faustino and
P. Puente-Payo
R. Marcos*
Affiliation:
Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
C. Pereira
Affiliation:
Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
J.P. Maia
Affiliation:
CIBIO Research Centre in Biodiversity and Genetic Resources, InBIO, University of Porto, Vila do Conde, PortugalBiology Department, Faculty of Sciences, University of Porto, Porto, Portugal
M. Santos
Affiliation:
Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
C. Luzzago
Affiliation:
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy
S. Lauzi
Affiliation:
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy
C. Genchi
Affiliation:
Department of Veterinary Science and Public Health, University of Milan, Milan, Italy
A. Faustino
Affiliation:
Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
P. Puente-Payo
Affiliation:
Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
*
*E-mail: rmarcos@icbas.up.pt
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Abstract

The prevalence of canine Dirofilaria infection in Maio Island (Cape Verde) was analysed by serology, morphological and molecular identification of the parasite species. Blood and sera were collected from 150 dogs and 80 cats aged over 6 months from various localities of the island. DNA was extracted from blood and samples were screened by polymerase chain reaction (PCR) using microfilaria-specific primers. No Dirofilaria immitis was found in dogs while D. repens microfilariae were found in 5.3% of dogs and 6% were positive by PCR. The species identity was confirmed by sequencing of PCR products, which showed almost 100% homology with D. repens European sequences published in GenBank. No difference in Dirofilaria infection was observed between males and females or in dogs with different weights. However, older dogs and those from the western part of Maio Island were more frequently infected. No Dirofilaria was found in cats. This study represents the first evidence of D. repens in Cape Verde (West Africa) and highlights the need for implementing control measures and for a better surveillance of dirofilariosis in Africa.

Information

Type
Short Communications
Information
Journal of Helminthology , Volume 91 , Issue 1 , January 2017, pp. 87 - 90
Copyright
Copyright © Cambridge University Press 2016 

Introduction

Canine and feline Dirofilaria infections are worldwide diseases, with a variable prevalence depending on the canine population, the presence of mosquito vectors and the climate (reviewed by McCall et al., Reference McCall, Genchi, Kramer, Guerrero and Venco2008). Global warming, pet travelling and relocation and adaptation of new vector species have dictated an expansion of the disease in recent years (Genchi et al., Reference Genchi, Rinaldi, Cascone, Mortarino and Cringoli2005; Simón et al., Reference Simón, Siles-Lucas, Morchón, González-Miguel, Mellado, Carretón and Montoya-Alonso2012). However, the rate of infection is largely unknown in some parts of the globe, such as in Africa (Schwan, Reference Schwan2009; Siwila et al., Reference Siwila, Mwase, Nejsum and Simonsen2015).

Islands may be viewed as natural laboratories for epidemiological studies, with a defined population of definitive hosts and vectors, in which significant samples can be managed. Maio Island (15°N, 23°W) is located in the Cape Verde archipelago (West Africa), 1500 km south-west from the Canary islands and 600 km west from Senegal. This island has a characteristic flat surface, with an area of 269 km2, with many inhabited settlements near the coast, and an estimated population of 530 dogs, 350 cats and 6740 humans (Garcia, Reference Garcia2010; Antunes, Reference Antunes2013). It is a well-known tourist destination, where many Europeans have set up second homes, and pet travelling is frequent. Interestingly, a case of Dirofilaria immitis infection was recently diagnosed on Maio Island in a dog with generalized adenomegaly and poor body condition (Pereira et al., Reference Pereira, Almeida, Malta, Vilaça and Payo-Puente2013). In the present study a survey of Dirofilaria infections in dogs and cats was undertaken for the first time on Maio Island.

Materials and methods

Collection and examination of dogs and cats

Dogs and cats were included in this study, applying a non-probabilistic sampling method. Ten localities were sampled in the western (Calheta, Morro and Porto Inglês) and eastern (Barreiro, Figueira da Horta, Ribeira de D. João, Pilar Cão, Alcatraz, Pedro Vaz and Praia Gonçalo) parts of the island. The number of dogs and cats sampled per locality ranged from 5 to 42 and from 1 to 20, respectively. They were brought by their owners, and criteria for inclusion were: (1) the owner consented to participate in the study; (2) autochthonous origin; (3) age over 6 months; (4) no previous treatment with preventative drugs currently used against canine and feline Dirofilaria infections. Clinically healthy dogs and cats were sampled in July and November 2012, respectively.

After physical examination, 2 ml of blood were collected in EDTA by jugular venipuncture and samples were preserved at 4°C until processing (up to 12 h). In the case of dogs, serum was also collected and stored at − 20°C for subsequent serological analysis. In addition, for each animal, 4 ×  30 μl of blood were spotted on to Whatman® filter paper in separate dots, which were dried and stored for molecular testing.

A buffy coat smear (BCS) was carried out for each animal, stained with Hemacolor (Merck, Darmstad, Germany) and screened by microscopy (Olympus, Tokyo, Japan CX31, at 100 ×  magnification) for the presence of microfilariae. Their morphology was checked according to Liotta et al. (Reference Liotta, Sandhu, Rishniw and Bowman2013).

In dogs, sera were analysed by a D. immitis antigen commercial test kit (Witness® HW, Synbiotics Corporation, Missouri, USA) with a sensitivity of 71% and specificity of 94% (Courtney & Cornell, Reference Courtney and Cornell1990), following the manufacturer's instructions.

DNA from dog blood samples was extracted using a commercial kit (NucleSpin® Tissue, Macherey-Nagel, Germany) (Silbermayr et al., Reference Silbermayr, Eigner, Duscher, Joachim and Fuehrer2014). For extraction of DNA from cat blood samples, standard high-salt methods were used (Sambrook et al., Reference Sambrook, Fritsch and Maniatis1989; Maia et al., Reference Maia, Harris, Carranza and Gómez-Díaz2014). Samples were screened with filarial genus-specific primers COIintF (5′-TGATTGGTGGTTTTGGTAA-3′) and COIintR (5′-ATAAGTACGAGTATCAATATC-3′) targeting the cytochrome c oxidase subunit 1 mitochondrial region (COI) of all filarial species (Casiraghi et al., Reference Casiraghi, Anderson, Bandi, Bazzocchi and Genchi2001, Reference Casiraghi, Bain, Guerrero, Martin, Pocacqua, Gardner, Franceschi and Bandi2004). Negative and positive controls were run with each reaction. Only samples confirmed through sequencing were considered positive. The identity of the sequences was confirmed to the species level using the BLAST algorithm on GenBank (Altschul et al., Reference Altschul, Gish, Miller, Myers and Lipman1990) and uncorrected p-distances were calculated in MEGA v. 6.06 (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). Sequences were deposited in GenBank under the accession numbers KT901783–KT901791.

Data analysis

Pearson's chi-square (χ2) and Fisher's exact tests were used to evaluate the differences between proportions of infected dogs and cats regarding sex, age, weight and island locality. Statistical comparisons were carried out using SPSS 15 software (IBM, Armonk, New York, USA), taking P <  0.05 as significant.

Results

One hundred and fifty dogs and 80 cats were sampled, which represented roughly a quarter of the animal population of the island. Of 150 dogs examined, 66.7% were males and 33.3% females; 46.0% were less than 2 years old, 39.3% were aged 2–4 years and 14.7% were more than 4 years old. Regarding weight, 38.0% weighed less than 10 kg, 42.0% weighed between 10 and 20 kg and 20.0% weighed more than 20 kg. Of the 80 cats, 52.5% were males and 47.5% females; 33.8% were less than 2 years old, 56.3% were aged 2–4 years and 10.0% were older than 4 years (weight was not recorded for cats). Similar numbers of dogs and cats were sampled in the eastern and western parts of the island.

Eight dogs (6 males and 2 females) were positive for D. repens microfilariae. The dogs were mostly from the west of Maio (4 in Calheta and 3 in Morro) with a single case in the eastern part (from Ribeira D. João). Overall prevalence of microfilaraemia was 5.3% (95% confidence interval (CI): 2.3–8.4)). Nine dogs were PCR-positive (prevalence 6%: CI 2.8–9.2), corresponding to the cases positive on BCS and an additional one (from Calheta). The COI sequences were 100% identical using the BLAST algorithm on GenBank for D. repens reported by Pascucci et al. (Reference Pascucci, Fico, DÁngelo, Serini and Cammà2007) and Suzuki et al. (Reference Suzuki, Kobayashi, Okata, Matsuzaki, Mori, Chen and Iwata2015), and differed less than 0.25% from other European sequences (Ferri et al., Reference Ferri, Barbuto, Bain, Galimberti, Uni, Guerrero, Ferte, Bandi, Martin and Casiraghi2009; Otranto et al., Reference Otranto, Brianti, Dantas-Torres, Weigl, Latrofa, Gaglio, Cauquil, Giannetto and Bain2011). All dogs were negative for D. immitis antigen.

No differences were found for sex (P= 1), or weight (P= 0.19). Regarding age, older dogs (>2 years old) were more frequently infected (9.9% vs. 1.4%). No differences were found between Maio localities (P= 0.36), but the prevalence of infection was higher in dogs resident in the west of the island (10.5%: CI 3.6–14.4) vs. dogs in the eastern part (1.3%: CI 0–3.9) (P= 0.016). Analysing the PCR results, the test had 88.9% sensitivity (CI 68.4–100) with 100% (CI 100) specificity. The positive predictive value of BCS was also 100% (CI 100), whereas the negative predictive value was 99.3% (CI 97.9–100). All cats sampled were negative for both circulating microfilariae and molecular analyses.

Discussion

Reports of D. immitis from some West African countries, including Senegal, Sierra Leone, Nigeria, Gabon and, more recently, Zambia (Siwila et al., Reference Siwila, Mwase, Nejsum and Simonsen2015) have been published. However, D. repens has never been identified. Previous cases of D. repens in Africa were restricted to South Africa and Mozambique (Schwan, Reference Schwan2009). In this way, this is the first report of D. repens in West Africa and the Macaronesian islands. Despite a previous report of a D. immitis-infected dog in Maio (Pereira et al., Reference Pereira, Almeida, Malta, Vilaça and Payo-Puente2013), in the present survey no dog was found to be positive. The only study in the Cape Verde archipelago, reported no Dirofilaria in Santiago Island, 20 km from Maio (Kirchner et al., Reference Kirchner, Brunner, Edelhofer and Joachim2008). It is noteworthy that in other Macaronesian islands, such as Madeira and the Canary Islands, mostly D. immitis was recorded (Montoya-Alonso et al., Reference Montoya-Alonso, Carretón, Corbera, Juste, Mellado, Morchón and Simón2011; Pantchev et al., Reference Pantchev, Etzold, Daugschies and Dyachenko2011; Cardoso et al., Reference Cardoso, Mendão and Madeira de Carvalho2012). Considering that the two species have the same intermediate hosts and extrinsic development (Simón et al., Reference Simón, Siles-Lucas, Morchón, González-Miguel, Mellado, Carretón and Montoya-Alonso2012) and that the average temperature is similar in all those islands, we can hypothesize that the spreading of the two species into Macaronesian islands has been due to the introduction of infected dogs, in which one or the other species was more prevalent. In the present survey, it is possible that cases of D. immitis might have been missed due to a low prevalence. Even though the examined sample covered about a quarter of the dog population of the island, we cannot exclude this. Furthermore, this survey was carried out with healthy dogs and, while D. immitis is well known to cause severe clinical symptoms, most D. repens infections run unnoticed (McCall et al., Reference McCall, Genchi, Kramer, Guerrero and Venco2008). It is likely that D. repens has been introduced from continental Europe, presumably by pet travelling, since the strains isolated in this survey were genetically identical to sequences from European cases (Pascucci et al., Reference Pascucci, Fico, DÁngelo, Serini and Cammà2007; Suzuki et al., Reference Suzuki, Kobayashi, Okata, Matsuzaki, Mori, Chen and Iwata2015). We are not able to state whether the disease was introduced recently or has been present for a long time on Maio Island. Nevertheless, the transmission cycle is likely to be established, due to the presence of dogs acting as reservoirs, competent vectors such as Aedes caspius and Culex pipiens mosquitoes (Alves et al., Reference Alves, Gomes, Rodrigues, Silva, Arez, Pinto and Sousa2010), and environmental conditions suitable for sustaining and expanding the infection. Additionally, the Cape Verde climate, with an average annual temperature of 24°C (Garcia, Reference Garcia2010), should allow the extrinsic development of D. repens all year round. The western part of the island showed a higher prevalence of infection and this is a relevant finding, considering the zoonotic relevance of D. repens (Simón et al., Reference Simón, Siles-Lucas, Morchón, González-Miguel, Mellado, Carretón and Montoya-Alonso2012) in an area where more than two-thirds of Maio's human population live (Garcia, Reference Garcia2010).

In conclusion, our data showed that canine D. repens infection is present in Maio Island. Although the current prevalence of microfilaraemic dogs is low (5.3%), it seems advisable to control and treat dogs, both living in and entering the island, with preventative drugs against the parasite. This would prevent the spreading of infection, reducing animal transmission, and would control the potential risk of human infections. For D. repens, repeated administrations of a spot-on formulation containing imidacloprid and moxidectin (Genchi et al., Reference Genchi, Genchi, Petry, Kruedewagen and Schaper2013; Petry et al., Reference Petry, Genchi, Schmidt, Schaper, Lawrenz and Genchi2015) or a sustained-release injectable formulation of moxidectin (Genchi et al., Reference Genchi, Prego and Genchi2010) have shown good results. The latter should be given every 6 months, to grant year-long protection. Alternatively, the use of subcutaneous doramectin (Baneth et al., Reference Baneth, Volansky, Anug, Favia, Bain, Goldstein and Harrus2002) could be a more cost-effective solution on Maio Island, where veterinary resources and medications are limited. Considering our data, studies assessing the prevalence of dirofilariosis in the other islands of the Cape Verde archipelago should be encouraged.

Conflict of interest

None.

Ethical standards

The authors assert that all procedures contributing to this work comply with ethical standards of the relevant national and institutional guides on the care and use of animals. The study was approved by local authorities, being included in an animal welfare campaign (Veterinários sem fronteiras (VETAID)).

References

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.CrossRefGoogle ScholarPubMed
Alves, J., Gomes, B., Rodrigues, R., Silva, J., Arez, A.P., Pinto, J. & Sousa, C.A. (2010) Mosquito fauna on the Cape Verde Islands (West Africa): an update on species distribution and a new finding. Journal of Vector Ecology 35, 307–312.Google Scholar
Antunes, A.C.L. (2013) Estimation and characterization of the dog and cat population on Maio island, Cape Verde: an integration of household survey data and remote sensing imagery. MSc thesis, Faculty of Veterinary Medicine, University of Lisbon.Google Scholar
Baneth, G., Volansky, Z., Anug, Y., Favia, G., Bain, O., Goldstein, R.E. & Harrus, S. (2002) Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectine. Veterinary Parasitology 105, 173–178.CrossRefGoogle Scholar
Cardoso, L., Mendão, C. & Madeira de Carvalho, L. (2012) 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. Parasites & Vectors 5, 62.Google Scholar
Casiraghi, M., Anderson, T.J., Bandi, C., Bazzocchi, C. & Genchi, C. (2001) A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122, 93–103.CrossRefGoogle ScholarPubMed
Casiraghi, M., Bain, O., Guerrero, R., Martin, C., Pocacqua, V., Gardner, S.L., Franceschi, A. & Bandi, C. (2004) Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution. International Journal of Parasitology 34, 191–203.Google Scholar
Courtney, C.H. & Cornell, J.A. (1990) Evaluation of heartworm immunodiagnostic tests. Journal of the American Veterinary Medical Association 197, 724–729.Google Scholar
Ferri, E., Barbuto, M., Bain, O., Galimberti, A., Uni, S., Guerrero, R., Ferte, H., Bandi, C., Martin, C. & Casiraghi, M. (2009) Integrated taxonomy: traditional approach and DNA barcoding for the identification of filaroid worms and related parasites (Nematoda). Frontiers in Zoology 6, 1.Google Scholar
Garcia, E.M.O. (2010) Gestão integrada dos recursos hídricos da ilha do Maio (Cabo Verde): o contributo das águas subterrâneas. MSc Thesis, Faculty of Sciences, University of Minho.Google Scholar
Genchi, C., Rinaldi, L., Cascone, C., Mortarino, M. & Cringoli, G. (2005) Is heartworm disease really spreading in Europe? Veterinary Parasitology 133, 137–148.Google Scholar
Genchi, M., Prego, G. & Genchi, C. (2010) Efficacy of moxidectin microsphere sustained release formulation for the prevention of subcutaneous filarial (Dirofilaria repens) infection in dogs. Veterinary Parasitology 170, 167–169.CrossRefGoogle ScholarPubMed
Genchi, C., Genchi, M., Petry, G., Kruedewagen, E.M. & Schaper, R. (2013) Evaluation of the efficacy of imidacloprid 10%/moxidectin 2.5% (Advocate®, Advantage® Multi, Bayer) for the prevention of Dirofilaria repens infection in dogs. Parasitology Research 112, 81–89.Google Scholar
Kirchner, M., Brunner, A., Edelhofer, R. & Joachim, A. (2008) Vector-borne parasites of dogs on the islands of Cabo Verde. Wiener Klinische Wochenschrift 120, 49–53.CrossRefGoogle ScholarPubMed
Liotta, J.L., Sandhu, G.K., Rishniw, M. & Bowman, D. (2013) Differentiation of the microfilariae of Dirofilaria immitis and Dirofilaria repens in stained blood films. Journal of Parasitology 99, 421–425.Google Scholar
Maia, J.P., Harris, D.J., Carranza, S. & Gómez-Díaz, E. (2014) A comparison of multiple methods for estimating parasitemia of hemogregarine hemoparasites (Apicomplexa: Adeleorina) and its application for studying infection in natural populations. PloS One 9, e95010.Google Scholar
McCall, J.W., Genchi, C., Kramer, L.H., Guerrero, J. & Venco, L. (2008) Heartworm disease in animals and humans. Advances in Parasitology 66, 193–285.Google Scholar
Montoya-Alonso, J.A., Carretón, E., Corbera, J.A., Juste, M.C., Mellado, I., Morchón, R. & Simón, F. (2011) Current prevalence of Dirofilaria immitis in dogs, cats and humans from the Island of Gran Canaria, Spain. Veterinary Parasitology 176, 291–294.Google Scholar
Otranto, D., Brianti, E., Dantas-Torres, F., Weigl, S., Latrofa, M.S., Gaglio, G., Cauquil, L., Giannetto, S. & Bain, O. (2011) Morphological and molecular data on the dermal microfilariae of a species of Cercopithifilaria from a dog in Sicily. Veterinary Parasitology 182, 221–229.Google Scholar
Pantchev, N., Etzold, M., Daugschies, A. & Dyachenko, V. (2011) Diagnosis of imported canine filarial infections in Germany 2008–2010. Parasitology Research 109, S61–S76.Google Scholar
Pascucci, I., Fico, R., DÁngelo, A.R., Serini, S. & Cammà, C. (2007) First notification in Italy of cardiopulmonary filarioses (heartworm disease) in a wolf (Canis lupus). Veterinaria Italiana 43, 851–858.Google Scholar
Pereira, P., Almeida, C., Malta, M., Vilaça, R. & Payo-Puente, P. (2013) First report of Dirofilaria immitis in the Republic of Cape Verde. Veterinary Parasitology 192, 290–291.Google Scholar
Petry, G., Genchi, M., Schmidt, H., Schaper, R., Lawrenz, B. & Genchi, C. (2015) Evaluation of the adulticidal efficacy of imidacloprid 10%/moxidectin 2.5% (w/v) spot-on (Advocate®, Advantage® Multi) against Dirofilaria repens in experimentally infected dogs. Parasitology Research 114, S131–S144.CrossRefGoogle ScholarPubMed
Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual. 545 pp. New York, Cold Spring Harbor Laboratory Press.Google Scholar
Schwan, E.V. (2009) Filariosis of domestic carnivores in Gauteng, KwaZulu-Natal and Mpumalanga provinces, South Africa, and Maputo Province, Mozambique. PhD Thesis, University of Pretoria, South Africa.Google Scholar
Silbermayr, K., Eigner, B., Duscher, G.G., Joachim, A. & Fuehrer, H.P. (2014) The detection of different Dirofilaria species using direct PCR technique. Parasitology Research 113, 513–516.CrossRefGoogle ScholarPubMed
Simón, F., Siles-Lucas, M., Morchón, R., González-Miguel, J., Mellado, I., Carretón, E. & Montoya-Alonso, J. (2012) Human and animal dirofilariasis: the emergence of a zoonotic mosaic. Clinical Microbiology Reviews 25, 507–544.Google Scholar
Siwila, J., Mwase, E.T., Nejsum, P. & Simonsen, P. (2015) Filarial infections in domestic dogs in Lusaka, Zambia. Veterinary Parasitology 210, 250–254.Google Scholar
Suzuki, J., Kobayashi, S., Okata, U., Matsuzaki, H., Mori, M., Chen, K.R. & Iwata, S. (2015) Molecular analysis of Dirofilaria repens removed from a subcutaneous nodule in a Japanese woman after a tour to Europe. Parasite 22, 21.Google Scholar
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30, 2725–2759.Google Scholar