Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran

  • Majid Khanmohammadi Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. and Department of Laboratory Science, Marand Branch, Islamic Azad University, Marand, Iran
  • Reza Falak Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran. and Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  • Ahmad Reza Meamar Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  • Mehdi Arshadi Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran, and Alzahra Hospitals Tabriz University of Medical Sciences, Tabriz, Iran
  • Lame Akhlaghi Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  • Elham Razmjou Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iranl Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran
Keywords: Wolbachia pipientis, Dirofilaria immitis, Cytochrome oxidase I (COI, Wolbachia surface protein (WSP), Phylogenetic analysis


Background: The purpose of this study was molecular detection and phylogenetic analysis of Wolbachia species of Dirofilaria immitis. Methods: Adult filarial nematodes were collected from the cardiovascular and pulmonary arterial systems of natural­ly infected dogs, which caught in different geographical areas of Meshkin Shahr in Ardabil Province, Iran, during 2017. Dirofilaria immitis genomic DNA were extracted.  Phylogenetic analysis for proofing of D. immitis was car­ried out using cytochrome oxidase I (COI) gene. Afterward, the purified DNA was used to determine the molecular pattern of the Wolbachia surface protein (WSP) gene sequence by PCR. Results: Phylogeny and homology studies showed high consistency of the COI gene with the previously-registered sequences for D. immitis. Comparison of DNA sequences revealed no nucleotide variation between them. PCR showed that all of the collected parasites were infected with W. pipientis. The sequence of the WSP gene in Wolbach­ia species from D. immitis was significantly different from other species of Dirofilaria as well as other filarial spe­cies. The maximum homology was observed with the Wolbachia isolated from D. immitis. The greatest distance be­tween WSP nucleotides of Wolbachia species found between D. immitis and those isolated from Onchocerca lupi. Conclusion: PCR could be a simple but suitable method for detection of Wolbachia species. There is a pattern of host specificity between Wolbachia and Dirofilaria that can be related to ancestral evolutions. The results of this phylogenetic analysis and molecular characterization may help us for better identification of Wolbachia species and understanding of their coevolution.


Kozek WJ, Rao RU. The discovery of Wolbachia in arthropods and nematodes–A historical perspective. Wolbachia: a bug's life in another bug. 5: Karger Publishers; 2007. p. 1-14.

Hertig M, Wolbach SB. Studies on Rickettsia-Like Micro-Organisms in Insects. The Journal of Medical Research. 1924;44(3):329-74.7.

Stouthamer R, Breeuwer JA, Hurst GD. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annual review of microbiology. 1999;53:71-102.

Teixeira L, Ferreira A, Ashburner M. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS biology. 2008;6(12):e2.

Karimi J, Darsouei R. Presence of the endosymbiont Wolbachia among some fruit flies (Diptera: Tephritidae) from Iran: A multilocus sequence typing approach. Journal of Asia-Pacific Entomology. 2014;17(1):105-12.

Slatko BE, Luck AN, Dobson SL, Foster JM. Wolbachia endosymbionts and human disease control. Molecular and biochemical parasitology. 2014;195(2):88-95.

Taylor MJ. Wolbachia in the inflammatory pathogenesis of human filariasis. Annals of the New York Academy of Sciences. 2003;990(1):444-9.

Slatko BE, Taylor MJ, Foster JM. The Wolbachia endosymbiont as an anti-filarial nematode target. Symbiosis. 2010;51(1):55-65.

Ferri E, Bain O, Barbuto M, Martin C, Lo N, Uni S, et al. New insights into the evolution of Wolbachia infections in filarial nematodes inferred from a large range of screened species. PloS one. 2011;6(6):e20843.

Martin C, Gavotte L. The bacteria Wolbachia in filariae, a biological Russian dolls’ system: new trends in antifilarial treatments. Parasite. 2010;17(2):79-89.

McHaffie J. Dirofilaria immitis and Wolbachia pipientis: a thorough investigation of the symbiosis responsible for canine heartworm disease. Parasitology research. 2012;110(2):499-502.

Grandi G, Quintavalla C, Mavropoulou A, Genchi M, Gnudi G, Bertoni G, et al. A combination of doxycycline and ivermectin is adulticidal in dogs with naturally acquired heartworm disease (Dirofilaria immitis). Veterinary parasitology. 2010;169(3):347-51.

Bazzocchi C, Mortarino M, Grandi G, Kramer LH, Genchi C, Bandi C, et al. Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. International Journal for Parasitology. 2008;38(12):1401-10.

Kramer L, Grandi G, Passeri B, Gianelli P, Genchi M, Dzimianski MT, et al. Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride. Veterinary parasitology. 2011;176(4):357-60.

McCall JW, Kramer L, Genchi C, Guerrero J, Dzimianski MT, Supakorndej P, et al. Effects of doxycycline on early infections of Dirofilaria immitis in dogs. Veterinary parasitology. 2011;176(4):361-7.

Kozek WJ. What is new in the Wolbachia/Dirofilaria interaction? Veterinary parasitology. 2005;133(2-3):127-32.

Saint Andre A, Blackwell NM, Hall LR, Hoerauf A, Brattig NW, Volkmann L, et al. The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science (New York, NY). 2002;295(5561):1892-5.

Brattig NW, Rathjens U, Ernst M, Geisinger F, Renz A, Tischendorf FW. Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes. Microbes and infection. 2000;2(10):1147-57.

Parvizi P, Fardid F, Soleimani S. Detection of a New Strain of Wolbachia pipientis in Phlebotomus perfiliewi transcaucasicus, a Potential Vector of Visceral Leishmaniasis in North West of Iran, by Targeting the Major Surface Protein Gene. Journal of arthropod-borne diseases. 2013;7(1):46-55.

Latrofa MS, Montarsi F, Ciocchetta S, Annoscia G, Dantas-Torres F, Ravagnan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasites & vectors. 2012;5:76.

Parvizi P, Bordbar A, Najafzadeh N. Detection of Wolbachia pipientis, including a new strain containing the wsp gene, in two sister species of Paraphlebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Memórias do Instituto Oswaldo Cruz. 2013;108(4):414-20.

Zhou W, Rousset F, O'Neil S. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proceedings of the Royal Society B: Biological Sciences. 1998;265(1395):509-15.

Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution. 1993;10(3):512-26.

Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular biology and evolution. 2016;33(7):1870-4.

Khedri J, Radfar MH, Borji H, Azizzadeh M, Akhtardanesh B. Canine Heartworm in Southeastern of Iran with Review of disease distribution. Iranian journal of parasitology. 2014;9(4):560-7.

Bokai S MA, Mohebali M, Hoseini H, Nadim A. Study on prevalence of dirofilariosis in Meshkinshahr-Northwest of Iran. J Fac Vet Med Tehran Univ.1(53):1-2.

Lefoulon E, Bain O, Makepeace BL, d'Haese C, Uni S, Martin C, et al. Breakdown of coevolution between symbiotic bacteria Wolbachia and their filarial hosts. PeerJ. 2016;4:e1840.

Bourtzis K. Wolbachia-based technologies for insect pest population control. Advances in experimental medicine and biology. 2008;627:104-13.

V DEPM, Mendes AM, Mauricio IL, Calado MM, Novo MT, Belo S, et al. Molecular detection of Wolbachia pipientis in natural populations of mosquito vectors of Dirofilaria immitis from continental Portugal: first detection in Culex theileri. Medical and veterinary entomology. 2016;30(3):301-9.

Dyab AK, Galal LA, Mahmoud AE, Mokhtar Y. Finding Wolbachia in Filarial larvae and Culicidae Mosquitoes in Upper Egypt Governorate. The Korean journal of parasitology. 2016;54(3):265-72.

Zha X, Zhang W, Zhou C, Zhang L, Xiang Z, Xia Q. Detection and characterization of Wolbachia infection in silkworm. Genetics and Molecular Biology. 2014;37(3):573-80.

Poorjavad N, Goldansaz SH, Machtelinckx T, Tirry L, Stouthamer R, van Leeuwen T. Iranian Trichogramma: ITS2 DNA characterization and natural Wolbachia infection. BioControl. 2012;57(3):361-74.

Tijsse-Klasen E, Braks M, Scholte EJ, Sprong H. Parasites of vectors--Ixodiphagus hookeri and its Wolbachia symbionts in ticks in The Netherlands. Parasites & vectors. 2011;4:228.

Bereczki J, Rácz R, Varga Z, Tóth JP. Controversial patterns of Wolbachia infestation in the social parasitic Maculinea butterflies (Lepidoptera: Lycaenidae). Organisms Diversity & Evolution. 2015;15(3):591-607.

Tabar MD, Altet L, Martinez V, Roura X. Wolbachia, filariae and Leishmania coinfection in dogs from a Mediterranean area. The Journal of small animal practice. 2013;54(4):174-8.

Simsek S, Ciftci AT. Serological and Molecular Detection of Dirofilaria Species in Stray Dogs and Investigation of Wolbachia DNA by PCR in Turkey. Journal of arthropod-borne diseases. 2016;10(4):445-53.

Rossi MI, Aguiar-Alves F, Santos S, Paiva J, Bendas A, Fernandes O, et al. Detection of Wolbachia DNA in blood from dogs infected with Dirofilaria immitis. Experimental parasitology. 2010;126(2):270-2.

Landum M, Ferreira CC, Calado M, Alho AM, Mauricio IL, Meireles JS, et al. Detection of Wolbachia in Dirofilaria infected dogs in Portugal. Veterinary parasitology. 2014;204(3-4):407-10.

Morchón R, Carretón E, Grandi G, González-Miguel J, Montoya-Alonso JA, Simón F, et al. Anti-Wolbachia Surface Protein Antibodies Are Present in the Urine of Dogs Naturally Infected with Dirofilaria immitis with Circulating Microfilariae But Not in Dogs with Occult Infections. Vector Borne and Zoonotic Diseases. 2012;12(1):17-20.

Enemark HL, Oksanen A, Chriél M, le Fèvre Harslund J, Woolsey ID, Al-Sabi MNS. Detection and molecular characterization of the mosquito-borne filarial nematode Setaria tundra in Danish roe deer (Capreolus capreolus). International Journal for Parasitology: Parasites and Wildlife. 2017;6(1):16-21.

Comandatore F, Cordaux R, Bandi C, Blaxter M, Darby A, Makepeace BL, et al. Supergroup C Wolbachia, mutualist symbionts of filarial nematodes, have a distinct genome structure. Open biology. 2015;5(12):150099.

Taylor MJ, Voronin D, Johnston KL, Ford L. Wolbachia filarial interactions. Cellular microbiology. 2013;15(4):520-6.

Armoo S, Doyle SR, Osei-Atweneboana MY, Grant WN. Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus. Parasites & vectors. 2017;10(1):188.

Belanger DH, Perkins SL. Wolbachia infection and mitochondrial diversity in the canine heartworm (Dirofilaria immitis). Mitochondrial DNA. 2010;21(6):227-33.

Bazzocchi C, Jamnongluk W, O'Neill SL, Anderson TJ, Genchi C, Bandi C. wsp gene sequences from the Wolbachia of filarial nematodes. Current microbiology. 2000;41(2):96-100.

Bandi C, Trees AJ, Brattig NW. Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases. Veterinary parasitology. 2001;98(1-3):215-38.

How to Cite
Khanmohammadi M, Falak R, Meamar AR, Arshadi M, Akhlaghi L, Razmjou E. Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran. J Arthropod Borne Dis. 13(1):83.
Original Article