Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica

Particle and Fibre Toxicology

Gad Baneth²  Víctor M Montenegro³  Diana Rojas¹  Alicia Rojas² 

[1] Departamento de Parasitología, Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica;Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot 76100, Israel;Laboratorio de Parasitología, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica

关键词: Costa Rica;    Knott’s test;    PCR;    Canine filariosis;    Cercopithifilaria bainae;    Acanthocheilonema reconditum;    Dirofilaria immitis;   

Others  :  1146233
DOI  :  10.1186/s13071-015-0783-8

 received in 2015-01-25, accepted in 2015-03-06,  发布年份 2015

【 摘 要 】

Background

Canine filarioids are important nematodes transmitted to dogs by arthropods. Diagnosis of canine filariosis is accomplished by the microscopic identification of microfilariae, serology or PCR for filarial-DNA. The aim of this study was to evaluate a molecular assay for the detection of canine filariae in dog blood, to compare its performance to other diagnostic techniques, and to determine the relationship between microfilarial concentration and infection with other vector-borne pathogens.

Methods

Blood samples from 146 dogs from Costa Rica were subjected to the detection of canine filarioids by four different methods: the microhematocrit tube test (MCT), Knott’s modified test, serology and a high resolution melt and quantitative real-time PCR (HRM-qPCR). Co-infection with other vector-borne pathogens was also evaluated.

Results

Fifteen percent of the dogs were positive to Dirofilaria immitis by at least one of the methods. The HRM-qPCR produced distinctive melting plots for the different filarial worms and revealed that 11.6% of dogs were infected with Acanthocheilonema reconditum. The latter assay had a limit of detection of 2.4x10⁻⁴ mf/μl and detected infections with lower microfilarial concentrations in comparison to the microscopic techniques and the serological assay. The MCT and Knott’s test only detected dogs with D. immitis microfilaremias above 0.7 mf/μl. Nevertheless, there was a strong correlation between the microfilarial concentration obtained by the Knott’s modified test and the HRM-qPCR (r = 0.906, p < 0.0001). Interestingly, one dog was found infected with Cercopithifilaria bainae infection. Moreover, no association was found between microfilaremia and co-infection and there was no significant difference in microfilarial concentration between dogs infected only with D. immitis and dogs co-infected with Ehrlichia canis, Anaplasma platys or Babesia vogeli.

Conclusions

This is the first report of A. reconditum and C. bainae in Costa Rica and Central America. Among the evaluated diagnostic techniques, the HRM-qPCR showed the most sensitive and reliable performance in the detection of blood filaroids in comparison to the Knott’s modified test, the MCT test and a serological assay.

【 授权许可】

   
2015 Rojas et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Otranto D, Dantas-Torres F, Brianti E, Traversa D, Petrić D, Genchi C et al.. Vector-borne helminths of dogs and humans in Europe. Parasit Vectors. 2013; 6:16. BioMed Central Full Text
• [2]López J, Valiente-Echeverría F, Carrasco M, Mercado R, Abarca K. Identificación morfológica y molecular de filarias canina en una comunidad semi-rural de la Región Metropolitana de Chile. Rev Chilena Infectol. 2012; 29:284-9.
• [3]Brown HE, Harrington LC, Kaufman PE, McKay T, Bowman DD, Nelson CT et al.. Key factors influencing canine heartworm, Dirofilaria immitis, in the United States. Parasit Vectors. 2012; 5:245. BioMed Central Full Text
• [4]Scorza AV, Duncan C, Miles L, Lappin MR. Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica. Vet Parasitol. 2011; 183:178-83.
• [5]Beaver PC, Brenes R, Vargas Solano G. Zoonotic filaria in a subcutaneous artery of a child in Costa Rica. Am J Trop Med Hyg. 1984; 33:583-5.
• [6]Brenes R, Beaver PC, Monge E, Zamora L. Pulmonary dirofilariasis in a Costa Rican man. Am J Trop Med Hyg. 1985; 34:1142-3.
• [7]Beaver PC, Brenes R, Ardon J. Dirofilaria from the index finger of a man in Costa Rica. Am J Trop Med Hyg. 1986; 35:988-90.
• [8]Rodríguez B, Arroyo R, Caro L, Orihel TC. Human dirofilariasis in Costa Rica. A report of three new cases of Dirofilaria immitis infection. Parasite. 2002; 9:193-5.
• [9]Rodríguez B, Ros-Alvarez T, Grant S, Orihel TC. Human dirofilariasis in Costa Rica: Dirofilaria immitis in periorbital tissues. Parasite. 2003; 10:87-9.
• [10]Magnis J, Lorentz S, Guardone L, Grimm F, Magi M, Naucke TJ et al.. Morphometric analyses of canine blood microfilariae isolated by the Knott’s test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis. Parasit Vectors. 2013; 6:48. BioMed Central Full Text
• [11]Hoch H, Strickland K. Canine and feline dirofilariasis: life cycle, pathophysiology, and diagnosis. Compend Contin Educ Vet. 2008; 30:133-40.
• [12]Rishniw M, Barr SC, Simpson KW, Frongillo MF, Franz M, Dominguez Alpizar JL. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol. 2006; 135:303-14.
• [13]Casiraghi M, Bazzocchi C, Mortarino M, Ottina E, Genchi C. A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris). Vet Parasitol. 2006; 141:368-72.
• [14]Latrofa MS, Dantas-Torres F, Annoscia G, Genchi M, Traversa D, Otranto D. A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes. Vet Parasitol. 2012; 185:181-5.
• [15]Wongkamchai S, Monkong N, Mahannol P, Taweethavonsawat P, Loymak S, Foongladda S. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay. Vector Borne Zoonotic Dis. 2013; 13:31-6.
• [16]Rojas A, Rojas D, Montenegro V, Gutierrez R, Yasur-Landau D, Baneth G. Vector-borne pathogens in dogs from Costa Rica: First molecular description of Babesia vogeli and Hepatozoon canis infections with a high prevalence of monocytic ehrlichiosis and the manifestations of co-infection. Vet Parasitol. 2014; 199:121-8.
• [17]Acuña P, Chávez A. Determinación de la prevalencia de Dirofilaria immitis en los distritos de San Martín de Porres, Rímac y Cercado de Lima. Rev Inv Vet Perú. 2002; 13:108-10.
• [18]Castillo A, Guerrero O. Técnica de concentración para microfilarias (en sangre). In: Técnicas de diagnóstico parasitológico. Castillo A, Guerrero O, editors. Editorial de Universidad de Costa Rica, San José, Costa Rica; 2006: p.74-5.
• [19]Abaxis Inc. VetScan Canine Heartworm Rapid Test. 2015. http://www.abaxis.com/veterinary/products/canine-heartworm-rapid-test.html. Accessed 18 Feb 2015.
• [20]Dantas-Torres F, Otranto D. Dirofilariosis in the Americas: a more virulent Dirofilaria immitis? Parasit Vectors. 2013; 6:288. BioMed Central Full Text
• [21]Bowman D, Little SE, Lorentzen L, Shields J, Sullivan MP, Carlin EP. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet Parasitol. 2009; 160:138-48.
• [22]Wei L, Kelly P, Ackerson K, Zhang J, El-Mahallawy HS, Kaltenboeck B et al.. First report of Babesia gibsoni in Central America and survey for vector-borne infections in dogs from Nicaragua. Parasit Vectors. 2014; 7:126. BioMed Central Full Text
• [23]Theis JH, Stevens F, Law M. Distribution, prevalence, and relative risk of filariasis in dogs from the State of Washington (1997–1999). J Am Anim Hosp Assoc. 2001; 37:339-47.
• [24]Reifur L, Thomaz-Soccol V, Montiani-Ferreira F. Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis. Vet Parasitol. 2004; 122:273-86.
• [25]Brianti E, Gaglio G, Napoli E, Giannetto S, Dantas-Torres F, Bain O et al.. New insights into the ecology and biology of Acanthocheilonema reconditum (Grassi, 1889) causing canine subcutaneous filariosis. Parasitology. 2012; 139:530-6.
• [26]Espírito-Santo MC, Alvarado-Mora MV, Pinto PL, de Brito T, Botelho-Lima L, Heath AR et al.. Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model. Exp Parasitol. 2014; 143:83-9.
• [27]Nakagaki K, Yoshida M, Nogami S. Experimental infection of Dirofilaria immitis in raccoon dogs. J Parasitol. 2007; 93:432-4.
• [28]Taylor AE. Maintenance of filarial worms in vitro. Exp Parasitol. 1960; 9:113-20.
• [29]Hou H, Shen G, Wu W, Gong P, Liu Q, You J et al.. Prevalence of Dirofilaria immitis infection in dogs from Dandong. China Vet Parasitol. 2011; 183:189-93.
• [30]Giangaspero A, Marangi M, Latrofa MS, Martinelli D, Traversa D, Otranto D et al.. Evidences of increasing risk of dirofilarioses in southern Italy. Parasitol Res. 2013; 112:1357-61.
• [31]McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. Adv Parasitol. 2008; 66:193-285.
• [32]Rawlings CA, Dawe DL, McCall JW, Keith JC, Prestwood AK. Four types of occult Dirofilaria immitis infection in dogs. J Am Vet Med Assoc. 1982; 180:1323-6.
• [33]Ionica AM, Matei IA, Mircean V, Dumitrache MO, D’Amico G, Gyorke A et al.. Current surveys on the prevalence and distribution of Dirofilaria spp. and Acanthocheilonema reconditum infections in dogs in Romania. Parasitol Res. 2015; 114:975-82.
• [34]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. BioMed Central Full Text
• [35]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.
• [36]Almeida GL, Vicente JJ. Cercopithifilaria bainae sp. n. parasita de Canis familiaris (L.) (Nematoda Filarioidea). Atas Soc Biol Rio de Janeiro. 1984; 24:18.
• [37]Otranto D, Brianti E, Dantas-Torres F, Weigl S, Latrofa MS, Gaglio G et al.. Morphological and molecular data on the dermal microfilariae of a species of Cercopithifilaria from a dog in Sicily. Vet Parasitol. 2011; 182:221-9.
• [38]Ionică AM, D’Amico G, Mitková B, Kalmár Z, Annoscia G, Otranto D et al.. First report of Cercopithifilaria spp. in dogs from Eastern Europe with an overview of their geographic distribution in Europe. Parasitol Res. 2014; 113:2761-4.
• [39]Cortes HC, Cardoso L, Giannelli A, Latrofa MS, Dantas-Torres F, Otranto D. Diversity of Cercopithifilaria species in dogs from Portugal. Parasit Vectors. 2014; 7:261. BioMed Central Full Text
• [40]Latrofa MS, Dantas-Torres F, Giannelli A, Otranto D. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis. 2014; 5:943-6.
• [41]Brianti E, Otranto D, Dantas-Torres F, Weigl S, Latrofa MS, Gaglio G et al.. Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae. Vet Parasitol. 2012; 183:330-7.