Detection of Dirofilaria spp. in Dogs by PCR

Bulletin UASVM, Veterinary Medicine 67(2)/2010 ISSN 1843-5270; Electronic ISSN 1843-5378 Detection of Dirofilaria spp. in Dogs by PCR Roberta CIOCAN, Gh. DĂRĂBUŞ, Olga JACSÓ, Éva FOK 1) Faculty of Veterinary Medicine, Department of Parasitology and Parasitic Diseases,, Romania; E-mail: roberta.ciocan@yahoo.com; 2) Szent István University, Faculty of Veterinary Science, Departament of Parasitology and Zoology, Budapest, Hungary. Abstract. Knowing that dirofilariosis is an emerging zoonosis, little known and studied in Romania, the aim of the study was to determine the prevalence of Dirofilaria spp. infection in dogs, in the western part of Romania. Since there is no epidemiological study on Dirofilaria spp. infection in dogs in Romania by molecular techniques, we consider opportune to examine the positive samples by this technique. Between February 2008 and August 2009 a number of 188 blood samples, from dogs in Timis and Hunedoara Counties, were collected. From the total amount of samples 96 were from Timis County and 92 were collected from Deva dog shelter, Hunedoara County. Blood samples were examination by different methods: Fresh blood smear, modified Knott's method, Diro-test Speed Diro/Heartworm (Bio Veto Test, France), ELISA technique ( D. immitis antigen) (EVL Netherlands). The positive blood samples with microfilariae were examined by PCR technique used for specific detection of D. immitis and D. repens described by (Casiraghi et al., 2006), at the Faculty of Veterinary Medicine Szent István, at Parasitology and Zoology Department, from Budapest, Hungary. All 8 dogs tested were positive for Dirofilaria repens infection. Keywords: Dirofilariosis, Dirofilaria immitis, Dirofilaria repens, dog, PCR, Romania INTRODUCTION Dirofilaria spp. is, in addition to Wucheria, Brugia, Onchocerca, Loa, Mansonella, Acanthocheilonema, Dipetalonema, a genus of Onchocercidae family. Is a part of Filaroidea suborder, order Spirurida, class Secernentea, subfilium Nematoda, phylum Nematodozoa, which together with Arthropoda form the Ecdysozoa kingdom. Most prevalent species in dogs and cats are Dirofilaria immitis and Diroflaria (Nochtiella) repens (Anderson, 2000; Horst, 2003). Six filarial species are known to cause the disease in humans: D. immitis, D. repens, D. striata, D. tenuis, D. ursi and D. spectans. Only two of them, Dirofilaria immitis and Dirofilaria repens occur occasionally in Europe (Horst, 2003). There are more than 70 species of mosquitoes in which microfilaria (first larval stage) can develop in stage 3 infective larvae (L3), and they were considered major vectors for the transmission of dirofilariosis. Involved genera are: Aedes, Anopheles, Culex, Culiseta, Mansonia and Psorophora (Otto and Jachowski, 1981). 40

MATERIALS AND METHODS The research was conducted at the Department of Parasitology and Parasitic Diseases of the Faculty of Veterinary Medicine, Romania and at the Faculty of Veterinary Medicine Szent István, at Parasitology and Zoology Department, from Budapest, Hungary. Between February 2008 and August 2009 a number of 188 blood samples from dogs in Timis and Hunedoara Counties, were collected. Dogs examined were cases of University Veterinary Clinics, at different s and aged between 7 months and 16 years. From Timis County came 96 blood samples and from Deva, Hunedoara County came 92 blood samples. All blood samples were examined by other methods. (Roberta Ciocan et al., 2009; 2010). Positive blood samples with microfilariae were examined using molecular techniques (PCR), at the Faculty of Veterinary Medicine Szent István from Budapest at Parasitology and Zoology Department, in 20-22 July 2009. The primers used for D. repens were 12SF 5'- ATG TTT TGA TTT TTT TGT AT-3' and for D. immitis were 12S 5' ATT TGT TGT AAT ATT ACG A-3' (BIO IDT). For DNA extraction from blood samples the Dneasy Blood & Tissue (QIAGEN SUA) kit was used. The PCR reaction with species specificity for detection of D. immitis and D. repens microfilariae, after (Casiraghi et al., 2006), was performed. DNA fragments obtained were analyzed using 1.5% agarose gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet light. PCR-products were sequenced directly using ABI technology to verify PCR specificity. RESULTS AND DISCUTIONS Dogs examined by PCR method Tab. 1 Locality Breed Age (years) Sex Fresh blood smear (microfilariae) Knott's Modified Diro- test (D. immitis) ELISA Kit (D. immitis) San Andrei Amstaff 7 M positive D. repens low positive negative D. repens PCR Ortisoara Ciarda Rosie Daniflor Deva Shelter Deva Shelter Caucazian Shepherd German Brak 8 F positive D. repens negative negative D. repens 9 F positive D. repens negative negative D. repens 4 M positive D. repens negative negative D. repens 12 M positive D. repens negative negative D. repens 2 M positive D. repens negative negative D. repens 3 M positive D. repens negative negative D. repens 5 F positive D. repens negative negative D. repens The eight dogs were positive for microfilariae in blood and modified Knott's method examination and in the test for antigens of D. immitis the results were negative. The Amstaff 41

patient, with a weak positive result at diro-test, was negative in ELISA test for D. immitis and positive at PCR tehnique for D. repens infection. By molecular techniques 8 samples were positive for Dirofilaria repens infection (Tab. 1). The dimensions of fragments amplified for 12S rdna fragments and coxi gene were approximately (400 bp). Fig. 1. Gel electrophoresis of filarial PCR products in a 1.5% agarose gel for the two species analyzed: D. immitis and D. repens (original). Legend: MSW standard molecular weight on a scale from 100 bp (amplicones); + positive control sample; - negative control sample; Dr: D. repens, 8 samples; Di: D. immitis, 8 samples. Top row are by products of DNA obtained by PCR with species specificity for D. repens and bottom row are by products of DNA obtained by PCR with species specific for D. immitis. It is noted that from the eight samples examined, five are strong positive: samples no: 2, 4, 5, 6, 7. Samples 1, 3 are medium positive and sample 8 is low positive (top row) (Fig. 1). At the DNA products obtained by PCR with species specificity for D. immitis can be observed that none of the samples is positive, only the positive control sample can be observed (bottom row) (Fig.1). Molecular technique used in this study allows easy and high specificity differentiation of the main filarial nematodes affecting dogs in Europe. This method can be very useful, especially since veterinary practitioners face more often increasingly complex issues of the nematodes morphology or doubtful results in serological tests (Genchi et al., 2005). The eight clinical cases were diagnosed 100% correctly by applying the species specificity of PCR technique described above, which demonstrates the high sensitivity of the 42

proposed technique. We have to note that none of the dogs infected with Dirofilaria spp, have left our country, all being autohtone cases. Dirofilariosis increased prevalence may be the consequence of the growth in stray canine population and lack of prevention. Infestation with Dirofilaria spp values, are influenced by mosquito population density and their species, but also the climatic and environmental variations (temperature, humidity, precipitation, vegetation and the presence of water courses) (Genchi et al., 2005). Dirofilariosis is a parasitosis common worldwide, often found in southern Europe, Spain, Portugal, France, Greece and Turkey. In Europe most endemic area is the Po river valley in northern Italy, where the prevalence of Dirofilaria immitis infecton in dogs is 40-80% and 24% in cats (Genchi et al., 2005; Kramer and Genchi, 2002). Cancrini et al. (2001), estimated an infection with D. repens of 37-85 % in dogs from Southern Spain. The distribution of Dirofilaria immitits infection in Spain is irregular. In the southern provinces of Huelva was 36.7%, Cadiz 12.0% and Badajoz 8.0% in 1991 (Cancrini, 2001). In the southern regions of Portugal, including Ribatejo the prevalence of D. immitis infection was 6.7%, Alentejo 16.5% and Algarve 12% in 1996 (Araujo, 1996). The prevalence of filarial infection in dogs from Greece was 10%, 30% and 8% for D. immitis, D. repens and Acanthocheilonema reconditum respectively, for the years 1987-1991 (Papazauhariadou et al., 1994). In Vojvodina region in Serbia, the prevalence of infection with D. repens was 49.22% in 2007 (Tasic et al., 2007). The prevalence of infection with D. immitis in Turkey in Kirikkale region, was 5.8% and the prevalence of occult infection was 27.46% in 2008 (Kander et al., 2008). In Bulgaria the prevalence of D. immitis infection was 8.62% between 2001 and 2006 (Zvezdelina Kirkova et al., 2007). Dirofilariosis prevalence in dogs in Belgrade was 22.01% for D. immitis and 19.26% for D. repens in the years 2007-2008 (Pavloć et al., 2009). Éva Fok et al. (2007), estimated an infection rate of 14% with D. repens in dogs in Hungary. CONCLUSIONS Only Dirofilaria repens was identified by molecular technique. Acknowledgements. Dr.Fok Evá and Dr. PhD Jacsó Olga from Faculty of Veterinary Medicine Szent István Budapest. REFERENCES 1. Anderson, R.C. (2000). Nematode parasites of vertebrates, their development and transmission. 2nd Edition, CABI Publishing, Wallingford, UK. 650. 2. Araujo, A.M. (1996). Canine and human Dirofilaria immitis infections in Portugal. A review. Parassitol. 38:366. 3. Cancrini, G., Kramer, L.H. (2001). Insect vectors of Dirofilaria spp. In: Simon F and Genchi C (Eds), Heartworm infection in humans and animals. Universidad Salamanca Ed. 63-82. 4. Casiraghi, M., Bazzocchi, C., Mortarino, M., Ottina, E., Genchi, C. (2006). A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris). Vet. Parasitol. 141:368-372. 43

5. Fok Evá (2007). The importance of dirofilariosis in carnivores and humans in Hungary, past and present. Present issue, Zagreb, Croaţia, First European Dirofilaria Days.183-188. 6. Genchi, C., Rinaldi, L., Cascone, C., Mortarino, M., Cringoli, G. (2005). Is heartworm disease really spreanding in Europe? Vet Parasitol. 133:137-148. 7. Genchi, C., Venco, L., Genchi, M. (2005). Guideline for the laboratory diagnosis of canine and feline Dirofilaria infections. Mappe Parassitologiche 8:139-144. 8. Hendrix, C.M., Robinson, E. (2006). Diagnostic parasitology for veterinary technicians, ed.3, St Louis, Mosby. 229. 9. Horst Aspöck (2003). Dirofilariae and dirofilarioses: Introductory remarks. Helminthologische Colloquium. 5. 10. Kander Yildiz., Sibel, D., Bugrahan, B., Yagci Naci, Ocal Ayca. (2008). The prevalence of Dirofilaria immitis in Dogs in Kirikkale, Turkish Society Parasitol. 32 (3):225-228. 11. Kramer, L., Genchi, C. (2002). Feline heartworm infection: serological survey of asymptomatic cats living in northern Italy, Vet Parasitol. 104:43-50. 12. Ortega-Mora, L.M., Gomez-Bautista. M., Rojo-Vazquez, F., Rodenas, A., Guerrero, J. (1991). A survey of the prevalence of canine filariasis in Spain. Preventive Vet Med. 11: 63-68. 13. Otto, G.F., Jachowski, J.R.L.A. (1981). Mosquitos and canine heartworm disease: In: GF Otto, ed. Proceedings of the Heartworm Symposium '80. Edwardsville, KS, Veterinary Medicine Publishing Co.17-32. 14. Papazauhariadou, M.G., Koutinas A.F., Rallis, T.S., Harailabidis S.T. (1994). Prevalence of microfilaremia in episodic weakness and clinically normal dogs belonging to hunting s, J. Helminthol. 68: 243 245. 15. Pavloć, I., Terzin, V., Ćurcin, L.J., Petković, D., Terzin, D., Ćurcin, K. (2009). Dirofilariosis prevalence at stray and pet dogs at Belgrade area in period 2007-2008. Scientific Veterinary Institute of Serbia, Belgrade, Vojvode Toze 14, Serbia. 2nd European Dirofilaria Days Salamanca, Spain. 227. 16. Roberta Ciocan, Dărăbuș, Gh., Ilie, M.S., Hotea Ionela, Morariu, S., Morar, D., Imre, K., Balint, A., Indre D. (2009). Preliminary observations an epidemiological survey in dirofilariosis of dogs from Timis County, Scientifical papers Veterinary Medicine 12(1):109-115. 17. Roberta Ciocan, Dărăbuș, Gh., Ilie, M.S., Mirela Imre, Ionela Hotea. (2010). Prevalence of dirofilariosis in dogs in Timis County, Scientifical papers Veterinary Medicine 13(1): 36-40. 18. Roberta Ciocan, Dărăbuș, Gh., Ilie, M.S.. Mirela Imre, Ionela Hotea. (2010). Dirofilaria repens infection prevalence in Hunedoara County, Scientifical papers Veterinary Medicine Iasi. 53. 19. Tasic, A., Suzana Tasic, Natasa Miladinovic, Dragan, Z., Jovana Djordjevic. (2007). Prevalence of Dirofilaria repens - cause of zoonose in dogs, Serbia. 20:71-74. 20. Zvezdelina Kirkova, Ivanov, A., Dimitrina Georgevina. (2007). Dirofilariosis in dogs and carnivores wild in Bulgaria, Zagreb, Croatia, First European Dirofilaria Days. 204. 44