Serological and molecular detection of Dirofilaria immitis in pet dogs of Lahore, Pakistan
DOI:
https://doi.org/10.17420/ap72.549Keywords:
heartworm disease, Dirofilaris immitis, parasitic nematodes, mosquitoes, dogs, microfilariaeAbstract
Heartworms, parasitic nematodes, are responsible to cause a potentially life threatening condition, heartworm disease in mammals particularly dogs and cats. The disease is prevalent in warm, humid areas with mosquitoes as intermediate hosts, increases the chances of transmission. This study aimed to investigate heartworm infection (Dirofilaria immitis) in pet dogs for the first time in Lahore using morphological, serological and molecular techniques including microscopy, PCR and rapid diagnostic kit tests. Blood samples were tested for antigens, with positive cases further analyzed using microscopy, Polymerase Chain Reaction, X-ray imaging, and echocardiography to detect heart-related changes. Modified Knott’s Test, remained a valuable tool for detecting and differentiating microfilariae, particularly when used alongside other diagnostic techniques. The microfilariae was confirmed morphologically through wet mount method but had low sensitivity and requires expertise, while microscopy showed high sensitivity but yielded more false positives compared to PCR. Thoracic radiography revealed characteristic signs of D. immitis infection, but may not show early-stage changes. PCR detected D. immitis in 10% of samples, with positive bands (at 203 bp for specific primers and 450 bp for general primers) observed for both species-specific and general primers. Using these diagnostic techniques improves early detection and treatment of heartworm disease in dogs, setting a new standard for veterinary care in Pakistan.
References
[1] Otranto D. 2018. Arthropod-borne pathogens of dogs and cats: from pathways and times of transmission to disease control. Veterinary Parasitology 251: 68–77. https://doi.org/10.1016/j.vetpar.2017.12.021
[2] Genchi C., Mortarino M., Rinaldi L., Cringoli G., Traldi G., Genchi M. 2011. Changing climate and changing vector-borne disease distribution: the example of Dirofilaria in Europe. Veterinary Parasitology 176(4): 295–299. https://doi.org/10.1016/j.vetpar.2011.01.012
[3] 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. https://doi.org/10.1016/S0065-308X(08)00204-2
[4] Hailu F.A., Tafesse G., Hailu T.A. 2020. Pathophysiology and gastrointestinal impacts of parasitic helminths in human beings. Journal of Pathology Research Reviews and Reports 2(2): 2–8. https://doi.org/10.47363/JPR/2020(2)122
[5] Che-Mendoza A., Guillermo-May G., Herrera- Bojórquez J., Barrera-Pérez M., Dzul-Manzanilla F., Gutierrez-Castro C., Arredondo-Jiménez J.I., Sánchez-Tejeda G., Vazquez-Prokopec G., Ranson H., Lenhart A., Sommerfeld J., McCall P.J., Kroeger A., Manrique-Saide P. 2015. Long-lasting insecticidetreated house screens and targeted treatment of productive breeding-sites for dengue vector control in Acapulco, Mexico. Transactions of the Royal Society of Tropical Medicine and Hygiene 109(2): 106–115. https://doi.org/10.1093/trstmh/tru189
[6] Genchi C., Venco L., Genchi M. 2007. Guideline for the laboratory diagnosis of canine and feline Dirofilaria infections. https://www.cabidigitallibrary.org/doi/pdf/10.5555/ 20083097549.
[7] Magnis J., Lorentz S., Guardone L., Grimm F., Magi M., Naucke T.J., Deplazes P. 2013. 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. Parasites & Vectors 6: 1–5. https://doi.org/10.1186/1756-3305-6-48
[8] Baticados W.N., Fernandez-Colorado C.P., Baticados A. 2011. Molecular detection of Trypanosoma evansi in cattle from Quirino Province, Philippines. Veterinarski Arhiv 81(5): 635–646. https://hrcak.srce.hr/file/108646.
[9] Hussain S., Hussain A., Aziz M.U., Song B., Zeb J., Hasib F.Y., Almendros A., Cabezas-Cruz A., George D., Sparagano O. 2023. First molecular confirmation of multiple zoonotic vector-borne diseases in pet dogs and cats of Hong Kong SAR. Ticks and Tick-borne Diseases 14(4): 102191. https://doi.org/10.1016/j.ttbdis.2023.102191
[10] 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 for Parasitology, 34(2): 191–203. https://doi.org/10.1016/j.ijpara.2003.10.004
[11] Torres-Chable O.M., Baak-Baak C.M., Cigarroa- Toledo N., Blitvich B.J., Brito-Argaez L.G., Alvarado-Kantun Y.N., Zaragoza-Vera C.V., Arjona- Jimenez G., Moreno-Perez L.G., Medina-Perez P. 2018. Molecular detection of Dirofilaria immitis in dogs and mosquitoes in Tabasco, Mexico. Journal of Vector-borne Diseases 55(2): 151–158. https://doi.org/10.4103/0972-9062.242563
[12] Roblejo-Arias L., Díaz-Corona C., Piloto-Sardiñas E., Díaz-Sánchez A.A., Zając Z., Kulisz J., Cabezas- Cruz A. 2023. First molecular characterization of Dirofilaria immitis in Cuba. BMC Veterinary Research 19(1): 239. https://doi.org/10.1186/s12917-023-03803-0
[13] Abou El-Naga T., Barghash S.M., Mohammed A.H.H., Ashour A.A., Salama M.S. 2012. Evaluation of (Rotat 1. 2-PCR) assays for identifying Egyptian Trypanosoma evansi DNA. Acta Parasitologica Globalis 3(1): 1–6. https://doi.org/10.5829/idosi.apg.2012.3.1.6681
[14] Bailén E.L., Martinez Gil C., Shorten E., O’Neill E.J. 2020. Case study: canine heartworm disease diagnosed in Ireland. Veterinary Ireland Journal 10(5): 251–256. https://www.veterinaryirelandjournal.com/images /pdf/focus/focus1_may_2020.pdf.
[15] Men S., Boutté Y., Ikeda Y., Li X., Palme K., Stierhof Y.D., Hartmann M.A., Moritz T., Grebe M. 2008. Sterol-dependent endocytosis mediates postcytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nature Cell Biology 10(2): 237–244. https://doi.org/10.1038/ncb1686
[16] Thompson J.D., Higgins D.G., Gibson T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22(22): 4673–4680. https://doi.org/10.1093/nar/22.22.4673
[17] Khan M.A., Shabir S., Azeem S., Gill W., Ashraf K., Azhar M., Rashid I., Ashraf M., Avais M., Ahmad A.S., Younas M., Badshah A., Ahmad S., Akbar H. 2023. Documentation of Trypanosoma evansi in captive tigers and lions in Punjab (2016–2018), Pakistan. Journal of Zoo and Wildlife Medicine 53(4): 823–831. https://doi.org/10.1638/2021-0053
[18] Iatta R., Sazmand A., Nguyen V.L., Nemati F., Ayaz M.M., Bahiraei Z., Zafari S., Giannico A., Greco G., Dantas-Torres F., Otranto D. 2021. Vector-borne pathogens in dogs of different regions of Iran and Pakistan. Parasitology Research 120(12): 4219–4228. https://doi.org/10.1007/s00436-020-06992-x
[19] Noack S., Harrington J., Carithers D.S., Kaminsky R., Selzer P.M. 2021. Heartworm disease – overview, intervention, and industry perspective. International Journal for Parasitology: Drugs and Drug Resistance 16: 65–89. https://doi.org/10.1016/j.ijpddr.2021.03.004
[20] Irwin P.J., Jefferies R. 2004. Arthropod-transmitted diseases of companion animals in Southeast Asia. Trends in Parasitology 20(1): 27–34. https://doi.org/10.1016/j.pt.2003.11.004
[21] Reifur L., Thomaz-Soccol V., Montiani-Ferreira F. 2004. Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis. Veterinary Parasitology 122(4): 273–286. https://doi.org/10.1016/j.vetpar.2004.05.017
[22] Trancoso T.A.L., Lima N.C., Barbosa A.S., Leles D., Fonseca A.B.M., Labarthe N.V., Bastos O.M.P., Uchôa C.M.A. 2020. Detection of Dirofilaria immitis using microscopic, serological and molecular techniques among dogs in Cabo Frio, RJ, Brazil. Revista Brasileira de Parasitologia Veterinária 29(1): e017219. https://doi.org/10.1590/S1984-29612020009
[23] Kotwa J.D., Jardine C.M., Pearl D.L., Berke O., Mercer N.J., Peregrine A.S. 2020. Evaluation of the SNAP® 4Dx® plus test for the detection of Dirofilaria immitis antigen and characterization of exposure to tick-borne pathogens in wild canids in southern Ontario. Veterinary Parasitology 283: 109176. https://doi.org/10.1016/j.vetpar.2020.109176
[24] Panarese R., Iatta R., Mendoza-Roldan J.A., Szlosek D., Braff J., Liu J., Beugnet F., Dantas-Torres F., Beall M.J., Otranto D.J.P. 2020. Comparison of diagnostic tools for the detection of Dirofilaria immitis infection in dogs. Pathogens 9(6): 499. https://doi.org/10.3390/pathogens9060499
[25] Girdan G.T., Anghel R.G., Ionita M., Mitrea I.L. 2015. Data on canine heartworm (Dirofilaria immitis) infection and other vector-borne pathogens in dogs in Bucharest area, Romania. Veterinary Medicine 61(1): 146–151.
[26] Khanmohammadi M., Akhlaghi L., Razmjou E., Falak R., Emameh R.Z., Mokhtarian K., Arshadi M., Tasbihi M., Meamar A.R. 2020. Morphological description, phylogenetic and molecular analysis of Dirofilaria immitis isolated from dogs in the Northwest of Iran. Iranian Journal of Parasitology 15(1): 57–66. https://pmc.ncbi.nlm.nih.gov/articles/PMC7244830/.
[27] Tudor N., Ionita L., Tapaloaga D., Tudor P., Ionita C., Vlagioiu C. 2014. Radiographic cardiopulmonary changes in dogs with heartworm disease. Romanian Biotechnological Letters 19(6): 9918–9924.
[28] Lombard C., Evans M., Martin L., Tehrani J. 1984. Blood pressure, electrocardiogram and echocardiogram measurements in the growing pony foal. Equine Veterinary Journal 16(4): 342–347. https://doi.org/10.1111/j.2042-3306.1984.tb01939.x
[29] Rishniw M., Barr S.C., Simpson K.W., Frongillo M.F., Franz M., Alpizar J.L.D. 2006. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Veterinary Parasitology 135(3–4): 303–314. https://doi.org/10.1016/j.vetpar.2005.10.013
