J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 83 http://jad.tums.ac.ir Published Online: April 27, 2019 Original Article Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran Majid Khanmohammadi1,2; Reza Falak3,4; Ahmad Reza Meamar1; Mehdi Arshadi1,5; *Lame Akhlaghi1; *Elham Razmjou1 1Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 2Department of Laboratory Science, Marand Branch, Islamic Azad University, Marand, Iran 3Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran 4Department of Immunology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 5Alzahra Hospitals', Tabriz University of Medical Sciences, Tabriz, Iran (Received 29 Mar 2018; accepted 26 Jan 2019) Abstract 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 natural- ly 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 car- ried 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 Wolbach- ia species from D. immitis was significantly different from other species of Dirofilaria as well as other filarial spe- cies. The maximum homology was observed with the Wolbachia isolated from D. immitis. The greatest distance be- tween 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. Keywords: Wolbachia pipientis; Dirofilaria immitis; Cytochrome oxidase I (COI); Wolbachia surface protein (WSP); Phylogenetic analysis Introduction Wolbachia is an intracellular α-proteobac- teria, endosymbiont in insects and filarial nem- atodes. This genus of bacteria belongs to the phylum of Proteobacteria and order of Rick- ettsiales. Wolbachia pipientis was the first bac- teria discovered in this genus, which infects Culex pipiens mosquito ovaries (1, 2) and plays a key role in the mosquito’s biology, ecology, immunity, evolution, parthenogenesis, microbi- al manipulation and reproduction (3, 4). This intracellular bacterium provides mechanisms to destroy male embryos and increase feminiza- tion of the filarial host, thus it is characterized by reproductive parasitism (3, 4). During fem- inization, these bacteria affect various biolog- ical characteristics including female worm fer- *Corresponding authors: Dr Lame Akhlaghi, E-mail: Akhlaghi.l@iums.ac.ir, Dr Elham Razmjou, E-mail: Razmjou.e@iums.ac.ir J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 84 http://jad.tums.ac.ir Published Online: April 27, 2019 tilization, molting, development, survival, and killing of the male hosts. Most likely, a cyto- plasmic incompatibility occurs and results in this phenomenon, however, up to now no de- finitive proof was found (4, 5). Wolbachia has a role in filarial nematode development, too. It mainly affects host’s re- productive system, increases the transfer of nematode larva from the insect to the mamma- lian host, affects embryogenesis, promotes pro- gression of the L1 form to the L3 form, reduc- es conversion time, and speeds up transition of the third-stage larva to adult forms (6). Wolbachia infection of the filarial nematodes may also cause mild inflammatory responses in mammalian hosts, too. This intracellular bac- terium is mostly located in the lateral hypoder- mal cords of both male and female nematodes. In this way, the worms may transmit their eggs to the cytoplasm of the female reproductive structures (7). Unfortunately, data regarding the symbiotic relationship between W. pipientis and D. immitis is limited; thus, the role of these bacteria in the pathogenesis of Dirofilaria, as the causative agent of canine heartworm dis- ease, is not fully understood. However, some data support the role of this bacterium in the pathophysiology and survival of distinct path- ogenic filarial worms including O. volvulus, W. bancrofti, B. malayi, D. repens, and D. im- mitis (8-10). Therefore, capability of this en- dosymbiont bacterium in manipulation of the reproductive system of the host could be ben- eficial for biological control and management of pests or for in molecular identification of the parasites. Wolbachia spp. could be a vaccine can- didate against vector-borne diseases including dengue and filariasis, too (3, 4). Antimicro- bial therapy against Wolbachia spp. may be useful in treatment of heartworm disease in dogs through decreasing the number of en- dogenous Wolbachia and consequently the microfilarial load, inhibition of the larval stage and also worm infertility. Typically, application of antimicrobial substances against Wolbachia may be useful as a complementary anti-filar- ial therapy strategy in canine and feline diro- filariasis, but further studies are needed to sub- stitute these novel methods for anti-parasite drugs (11). In this regard, doxycycline is an effective treatment against Wolbachia, and in combination with ivermectin has been shown to have adulticidal efficiency for heartworm treatments in dogs (12-15). Wolbachia met- abolic products may also exert pathological changes in many organs of the host such as lungs and kidneys (16). The Wolbachia surface protein (WSP) is a crucial component involved in the immuno- pathogenesis of filarial diseases and helps in parasite evasion from potentially harmful im- mune responses of the host (16). Dirofilaria immitis is a selective reservoir for W. pipientis, therefore, this bacterium provides attenuation strategy for Dirofilaria to evade host immune responses (17, 18). Wolbachia is effective in generating immune responses during heart- worm infections; thus, Wolbachia likely influ- ences the inflammatory and immunoregula- tory functions of the host (19). Therefore, its control may offer therapeutic and diagnostic possibilities. Elimination of Wolbachia by an- timicrobial agents can exert a preventive effect on embryogenesis of D. immitis and could have potential application in sterilization of the fe- male worms and consequently control and treat- ment of dirofilariasis (16). Interfering of this obligate relationship has been used in novel therapeutic strategies; for example, a combi- nation of antimicrobial therapy using doxycy- cline and ivermectin, as a macrocyclic lactone can destroy nematodes (8, 11, 14). Overall, due to the zoonotic potential and the increasing number of D. immitis and D. repens infections in non-endemic areas, xenomonitoring of this bacterium may be useful (20). In Iran, few studies have focused on detec- tion of Wolbachia species or their invertebrate hosts. Herein, we performed molecular detec- tion and phylogenetic analysis of Wolbachia in D. immitis, using the WSP gene. The main J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 85 http://jad.tums.ac.ir Published Online: April 27, 2019 reasons for selecting WSP gene in this study was the high availability of the corresponding protein in soluble and membranous forms in Wolbachia species, the feasibility of the am- plifying its coding sequence by PCR, con- siderable dissimilarity of its expression in dif- ferent filarial genus, and its applicability as an indicator of parasite evolution and bacterial phylogenic proximity (21). Materials and Methods Forty-three stray domestic dogs (Canis fa- miliaris) suspicious to dirofilariasis were col- lected during 2017 from different geograph- ical areas of Meshkinshahr, Ardebil Province, Iran. Meshkinshahr (38°23′56″N 47°40′55″E) is a main endemic area for canine dirofilariasis in Iran. All dogs were tested for dirofilariasis by direct microscopy, ELISA, and rapid dip- stick methods. Direct wet smears of whole blood were taken with EDTA and used for detection of microfilaria by light microscopy. Sera was analyzed for adult D. immitis circu- lating antigen using a commercial ELISA kit (DiroCHEK®, USA) as well as dipstick meth- od (SNAP® 4Dx® Test, CHW II kit IDEXX Laboratory, USA) according to the manufac- turers' instructions. All procedures were carried out in admis- sion with the rules and regulations of the re- spective national animal Ethics Committee of Iran University of Medical Sciences (IR. IUMS.REC1395.9221577203-2016.05.09(. Six hyper-infected dogs euthanized and necropsied. Worms were collected from the pulmonary arteries and right ventricles. Then, their DNA was extracted and used for molec- ular analyses. DNA extraction Adult worms were homogenized by a rotor- stator system (IKA, UK) and total genomic DNA was extracted using DNA extraction kit (QIAGEN GmbH, Germany) from approxi- mately 25mg of each Dirofilaria sample, ac- cording to the manufacturer’s instruction. Pu- rity of the extracted DNA was determined and DNA samples stored at -20 °C. For amplifica- tion of cytochrome oxidase I gene (COI) which is a species-specific mitochondrial gene of D. immitis, a set of specific primers were de- signed. In details, 5'-TGA TTG GTG GTT TTG GTA A-3' and 5'-ATA AGT ACG AGT ATC AATATC-3' were used as forward and reverse primers, respectively. In addition, we used Wolbachia-specific primers to amplify nucleotides 81–691 of WSP. The forward and reverse primers were 5' TGGTCCAATAAG TGATGAAGAAAC-3' and 5' AAAAATT AAACGCTACTCCA-3', respectively. Reac- tions were performed in 25μl volumes using 2x PCR Master Mix (RED Amplicon, Den- mark), 1μl of DNA template, and 1μl of each primer. The thermal cycler program included one cycle of 94 °C for 5min followed by 35 cycles of 94 °C for 1min (denaturation), 55 °C for 1min (annealing), 72 °C for 60sec (exten- sion), and a final extension at 72 °C for 10min (22). PCR products were electrophoresed on 1.5% agarose gel along with a commercial DNA marker (SMOBiO DM3100) using an imaging system (SYNGENE, UK). Finally, PCR products were purified using the PCR purification Kit (QIAGEN GmbH, Germany) and directly subject for sequencing. Molecular and phylogenetic analysis Phylogenetic analysis of D. immitis was carried out using COI gene sequences, obtained during the study (Accession code, MF288560.1 Mesh-Iran 3) along with previously determined relevant sequences in the GenBank. Then, all obtained nucleotide sequences were analyzed and compared with the available complete WSP sequence in GenBank (MG010709.1) using Bi- oEdit software, ver. 7.0.5 (California, USA). All of the evolutionary aspects and other mo- lecular data were inferred using the maximum likelihood method, based on the Tamura-Nei model (23). The phylogenetic tree was drawn to scale, with branch lengths measured as the J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 86 http://jad.tums.ac.ir Published Online: April 27, 2019 number of Wolbachia substitutions per site and with the highest log likelihood. Multiple alignments and sequences linking functional- ity of phylogenetic studies were analyzed with MEGA7 software (24). The accuracy of the phylogenetic tree was measured by 1000 boot- strap replication. Nucleotide changes were compared to the reference sequence with Se- quencher software (Sequencher® version 5.4.6 USA). Results We examined blood samples from 43 dogs with obvious symptoms of dirofilariasis and found that 27 dogs were Dirofilaria seroposi- tive (62.8% CI: 47.9 to 75.6). DNA was pu- rified from the collected worms and used as PCR template. Wolbachia DNA was detected in all 67 filarial worms including 41 females and 26 male parasites. Electrophoresis of PCR product of the amplified WSP gene confirmed presence of a 630bp Wolbachia specific sequence in all of the DNA extracts (Fig. 1). The BLASTn anal- ysis of the COI gene indicated maximum ho- mology of the isolated parasites’ DNA with previously isolated sequences of D. immitis. Phylogenetic analysis showed highest homol- ogy with D. immitis isolated from dogs in Ker- man City (KR 870344.1). We also found good homology with D. immitis isolated from sev- eral other geographical areas such as those dogs from Iran (KT960976.1, KT318126. 1), China (EU159111. 1), Italy (FN391553. 1) and Bangladesh (KC107805.1), and also other an- imals such as jackal (Canis aureus) in North Khorasan of Iran (KT351850.1, KT351851.1, KT351852. 1), cat from Iran (KT282097. 1) and Italy (AM749227. 1), wolf from Italy (DQ358815. 1) and red panda from China (EU169124.1) (Fig. 2). Sequence analysis in all samples demonstrated a DNA band which belonged to WSP gene of W. pipientis. Se- quence similarities and homologies between the amplified one and the previously regis- tered sequences in the GenBank was deter- mined following a basic local alignment se- quence analysis, and calculating the statisti- cal significance through the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequences were aligned with the online clustalW2 tool (http://www.ebi.ac.uk/Tools/msa/clustalw2). All obtained sequences were blasted after align- ment and belonged to W. pipientis. Partial D. immitis WSP nucleotide sequences were sub- mitted with accession code of MG010709.1 in GenBank. The phylogenetic analysis of WSP sequenc- es of Wolbachia showed maximum homology (99%) with the complete W. pipientis sequence isolated from D. immitis in Italy (AJ252062.1) and several other filarial organisms from dif- ferent areas of the world including D. repens (AJ252176.1), O. volvulus (HG810405.1), O. ochengi (HE660029.1), O. cervicalis (AY 095210.1), O. gibsoni (AJ252178.1), and O. gutturosa (AJ276497.1) (Fig. 3). Wolbachia pipientis is genetically related to the filarial nematodes phylum. Phylogenetic studies using WSP revealed the greatest simi- larity and proximity of the obtained sequence with other filarial nematodes. The least simi- larity of the Wolbachia sequence isolated from filarial nematodes was observed between On- chocerca and Dirofilaria taxa (Fig. 3). Nucleotide changes were compared and no differences were found between the amplified sequences and the previously reported ones. The highest sequence similarity with W. pipi- entis was seen in O. gibsoni and O. gutturosa, and the lowest was in O. lupi due to difference in 33 nucleotides. The nucleotide difference between D. immitis and D. repens was found in 27 nucleotides. The difference between D. repens and the other filarial nematodes were lowest of those studied ones (Fig. 4). All stud- ied species showed the greatest nucleotide dif- ferences between positions 143 and 161 of the known nucleotide location. This study showed https://blast.ncbi.nlm.nih.gov/Blast.cgi http://www.ebi.ac.uk/Tools/msa/clustalw2 J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 87 http://jad.tums.ac.ir Published Online: April 27, 2019 sequence variation in WSP gene of Wolbachia from various filarial nematode population. Fig. 1. Electrophoresis of PCR product of WSP specific amplification confirming the presence of endosymbiotic Wolbachia present in Dirofilaria immitis DNA extract. M: 100bp DNA marker, NC: Negative control, Lines 1–2: PCR products, PC: Positive control Fig. 2. Phylogenetic tree of Dirofilaria immitis cytochrome c oxidase subunit I (COI) gene using maximum likeli- hood analysis among sequences based on the Tamura-Nei model with 1000 bootstrap repetition. The scale bar indi- cates the genetic distance in single nucleotide substitutions. GenBank registered accession numbers are specified within parentheses. Ascaris lumbricoides (Accession no. AB591801.1) was as outgroup J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 88 http://jad.tums.ac.ir Published Online: April 27, 2019 Fig. 3. Phylogenetic tree of Wolbachia WSP gene sequences from Meshkinshahr of North West Iran and other pre- vious registered sequences from different areas using maximum likelihood method based on the Tamura-Nei model with 1000 bootstrap repetition Fig. 4. Multiple alignments of the partial WSP gene and the sequences of other filarial nematodes J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 89 http://jad.tums.ac.ir Published Online: April 27, 2019 Discussion This was the first report on molecular char- acterization of W. pipientis infection of D. im- mitis isolated from northwest of Iran, where, dirofilariasis is endemic. The prevalence of dirofilariasis in the untreated dogs was previ- ously estimated to be in the range of 1.4–51.4% (25, 26). Results of phylogenetic analysis of COI gene in the isolated filarial nematodes con- firmed that the Dirofilaria species in Kerman and Meshkinshahr are molecularly identical. Pairwise homology analysis revealed high se- quence homology of the isolated worms with the available sequences at NCBI GenBank. The determined Wolbachia infection rate was very similar to previous studies. The findings also confirmed the general believe about Wolbachia incidence and emphasized the absence of an- cestral infection in the nematode phylum (27). This widespread bacterium may be detect- ed in all classes of arthropods and approximate- ly infects 20–70% of them. Therefore, due to high incidence of the endosymbiosis infection of the arthropods with Wolbachia, recent stud- ies were mainly focused on arthropods (28). In brief, it is commonly found in butterflies, fruit flies, sand flies, mosquitoes, fleas, ticks, bugs, wasps, silkworms (4, 5, 19, 21, 29-34). Previ- ously, Wolbachia DNA was detected in 30.6% of the blood sample of filaria-positive dogs in the Mediterranean area (35). Co-infection of six dogs with D. immitis and D. repens was reported in Turkey by PCR-amplification of the WSP gene (36). Wolbachia DNA was also found in blood samples from Dirofilaria-in- fected dogs, in other regions (37). In a Por- tuguese study, dogs whose dirofilariasis was confirmed by parasitological and serological methods were further examined by PCR meth- od. Moreover, Wolbachia DNA was detected in blood sample of 52.6% of those with oc- cult infection. In occult infections, no obvious microfilaria could be found in microscopic ex- amination of blood. This molecular evaluation is useful because the presence of Wolbachia in association with microfilaria is critical for control of canine dirofilariasis (38). Anti-WSP IgG titer in urine samples from 19 Dirofilaria- infected dogs was greater than the established cut-off values, and WSP gene was detected in kidney glomerular capillaries in those that were seropositive, too (39). Wolbachia has also previously been isolat- ed from W. bancrofti, Brugia malayi, O. vol- vulus, and Setaria tundra (29, 40-42). Recent- ly, the study of the isolated O. volvulus from 4 African countries indicated that extensive intra species heterogeneity exists in the Wolbachia content of male and female adults of O. vol- vulus. Wolbachia variation was previously re- ported in some studies and was in line with our results and pointed out to the host speci- ficity of this bacterium (43). Diversity of the pattern of the mitochondrial genetic content was found in D. immitis and was much lower than the related species (44). Phylogenetic studies confirmed homology of the amplified sequence with a previously registered Wolbachia sequence in GenBank. We found the most consistency with Wolbach- ia species isolated from D. immitis in Italy (45). Filarial nematodes have gained this bacterium endosymbiosis from arthropods, and the current data indicates that this bacteria has spread glob- ally in filarial nematodes (46). We hypothesized that Wolbachia has a genetic relationship with the phylum of filarial nematodes and this as- sociation was most likely generated over times. Sequence comparisons between Dirofilaria species and other filarial nematodes revealed difference of several nucleotides in WSP gene. Compression of the sequences from our study with previously-reported Wolbachia sequences indicated that the amplified sequence is like- ly Dirofilaria-specific. Interestingly, BLASTn studies showed a specific WSP pattern in Diro- filaria. This could be due to genetic character- istics of the endosymbiont bacteria, as well as the physiological properties and adaptation of J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 90 http://jad.tums.ac.ir Published Online: April 27, 2019 Dirofilaria, and the regulatory mechanisms of the immune system of the final host. Many questions still remain unanswered in this field. Regarding to the reported distri- bution of Wolbachia in filarial nematodes, it is not clear whether the absence of W. pipi- entis in Wolbachia-negative filarial nematodes represents an ancestral characteristic of this bacteria or is due to losing the inserted sequence over time due to low transpositional activity in some filarial species (41). The phylogenies of various filarial nematodes are still not identi- fied completely; thus, it is difficult to express the presence or absence of W. pipientis on a tree indicating filarial genetic evolution (42). The presence of such extensive proprietary pattern between adult worms of both sexes in filarial nematodes provides questions regard- ing the specific symbiosis between the Wolbachia and filarial worms, and the mech- anisms by which Wolbachia is involved. Fi- nally, our results suggest that W. pipientis isolated from Iranian Dirofilaria has distinct genomic features, which likely are the result of long periods of independent evolution. The results of this study open the way for further studies on the strain identification and genet- ic diversity, which may increase our under- standing about the host-parasite relationships. Overall, the mapping of Wolbachia on the phy- logenetic trees generated by Mega software in- dicates that this bacterium may have evolved along with ancestors of filarial nematodes. Conclusion Main differences between the population of adult filarial worms were at the level of Wolbachia species. There is a pattern of host specificity between Wolbachia and Dirofilaria. This subject can be due to ancestral evolutions long times ago. Molecular characterization can be applied as a new trend for understanding the evolution and identification of Wolbachia. This method offers a new technique for diagnosis and may provide strategies for development of novel and effective therapeutic procedures. The results of this study can help us to under- stand the interactions of Wolbachia and Diro- filaria with their mammalian hosts. We sug- gest further investigation of the endosymbi- otic W. pipientis polymorphisms in nematodes. Findings also highlighted correlations between Wolbachia and D. immitis throughout their life cycle and could be a valuable resource that may be applied for providing novel intervention strategies, not only for the treatment and prophylaxis of dirofilariasis but also in other closely related human filarial disease as well. Acknowledgements The authors thank Professors Mehdi Mohe- bali and Iraj Mobedi for their valuable sug- gestions. Special thanks to Dr Zabiholah Zarei for his technical assistance and all colleagues in the Meshkinshahr Health Research Station. This study was financially supported by a grant from the Research Council of Iran Universi- ty of Medical Sciences (Project No, 27899). The authors declare that there is no con- flict of interests. References 1. Kozek WJ, Rao RU (2007) The discovery of Wolbachia in arthropods and nem- atodes–A historical perspective. Wolbach- ia: a bug's life in another bug. Karger Publishers. pp. 1–14. 2. Hertig M, Wolbach SB (1924) Studies on Rickettsia-Like Micro-Organisms in In- sects. J Med Med Res. 44(3): 329–374. 3. Stouthamer R, Breeuwer JA, Hurst GD (1999) Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu Rev Microbiol. 53: 71–102. 4. Teixeira L, Ferreira A, Ashburner M (2008) The bacterial symbiont Wolbachia induc- es resistance to RNA viral infections in J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 91 http://jad.tums.ac.ir Published Online: April 27, 2019 Drosophila melanogaster. PLoS Biol. 6 (12): e2. 5. Karimi J, Darsouei R (2014) Presence of the endosymbiont Wolbachia among some fruit flies (Diptera: Tephritidae) from Iran: A multilocus sequence typing ap- proach. J Asia Pac Entomol. 17(1): 105– 112. 6. Slatko BE, Luck AN, Dobson SL, Foster JM (2014) Wolbachia endosymbionts and human disease control. Mol Biochem Par- asitol. 195(2): 88–95. 7. Taylor MJ (2003) Wolbachia in the inflam- matory pathogenesis of human filariasis. Ann N Y Acad Sci. 990(1): 444–449. 8. Slatko BE, Taylor MJ, Foster JM (2010) The Wolbachia endosymbiont as an anti-filar- ial nematode target. Symbiosis. 51(1): 55–65. 9. Ferri E, Bain O, Barbuto M, Martin C, Lo N, Uni S (2011) New insights into the evolu- tion of Wolbachia infections in filarial nematodes inferred from a large range of screened species. PloS One. 6(6): e20843. 10. Martin C, Gavotte L (2010) The bacteria Wolbachia in filariae, a biological Rus- sian dolls’ system: new trends in antifi- larial treatments. Parasite. 17(2): 79–89. 11. McHaffie J (2012) Dirofilaria immitis and Wolbachia pipientis: a thorough inves- tigation of the symbiosis responsible for canine heartworm disease. J Parasitol Res. 110(2): 499–502. 12. Grandi G, Quintavalla C, Mavropoulou A, Genchi M, Gnudi G, Bertoni G (2010) A combination of doxycycline and ivermec- tin is adulticidal in dogs with naturally acquired heartworm disease (Dirofilaria immitis). Vet Parasitol. 169(3): 347–351. 13. Bazzocchi C, Mortarino M, Grandi G, Kra- mer LH, Genchi C, Bandi C (2008) Com- bined ivermectin and doxycycline treat- ment has microfilaricidal and adulticidal activity against Dirofilaria immitis in ex- perimentally infected dogs. Int J Parasi- tol. 38(12): 1401–1410. 14. Kramer L, Grandi G, Passeri B, Gianelli P, Genchi M, Dzimianski MT (2011) Eval- uation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combina- tion of doxycycline and ivermectin be- fore administration of melarsomine dihy- drochloride. Vet Parasitol. 4: 357–360. 15. McCall JW, Kramer L, Genchi C, Guerrero J, Dzimianski MT, Supakorndej P (2011) Effects of doxycycline on early infections of Dirofilaria immitis in dogs. Vet Par- asitol. 176(4): 361–367. 16. Kozek WJ (2005) What is new in the Wolbachia/Dirofilaria interaction. Vet Parasitol. 133(2–3): 127–132. 17. Saint Andre A, Blackwell NM, Hall LR, Hoerauf A, Brattig NW, Volkmann L (2002) The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science. 295(5561): 1892–1895. 18. Brattig NW, Rathjens U, Ernst M, Rathjens U, Ernst M, Geisinger F, Renz A, Tischendorf FW (2000) Lipopolysac- charide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate me- diators in the sequence of inflammatory and anti inflammatory responses of hu- man monocytes. Microb Infect. 2(10): 1147–1157. 19. Parvizi P, Fardid F, Soleimani S (2013) Detection of a New Strain of Wolbach- ia pipientis in Phlebotomus perfiliewi transcaucasicus, a potential vector of vis- ceral leishmaniasis in north west of Iran, by targeting the major surface protein gene. J Arthropod Borne Dis. 7(1): 46–55. 20. Latrofa MS, Montarsi F, Ciocchetta S, An- noscia G, Dantas-Torres F, Ravagnan S (2012) Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasit Vectors. 5: 76. J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 92 http://jad.tums.ac.ir Published Online: April 27, 2019 21. Parvizi P, Bordbar A, Najafzadeh N (2013) Detection of Wolbachia pipientis, in- cluding a new strain containing the wsp gene, in two sister species of Paraph- lebotomus sandflies, potential vectors of zoonotic cutaneous leishmaniasis. Mem Inst Oswaldo Cruz. 108(4): 414–420. 22. Zhou W, Rousset F, O'Neil S (1998) Phy- logeny and PCR-based classification of Wolbachia strains using wsp gene se- quences. Proc R Soc Lond (Biol). 265 (1395): 509–515. 23. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 512(3): 10–26. 24. Kumar S, Stecher G, Tamura K (2016) Mo- lecular evolutionary genetics analysis ver- sion 7.0 for bigger datasets. Mol Biol Evol. 33(7): 1870–1874. 25. Khedri J, Radfar MH, Borji H, Azizzadeh M, Akhtardanesh B (2014) Canine heart- worm in southeastern of Iran with re- view of disease distribution. Iran J Par- asitol. 9(4): 560–567. 26. Bokai S, Mohebali M, Hoseini H, Nadim A (1998) Study on prevalence of dirofi- lariosis in Meshkinshahr-Northwest of Iran. J Fac Vet Med Tehran Univ. 1(53): 1–2. 27. Lefoulon E, Bain O, Makepeace BL, d'Haese C, Uni S, Martin C (2016) Break- down of coevolution between symbiotic bacteria Wolbachia and their filarial hosts. Peer J. 4: e1840. 28. Bourtzis K (2008) Wolbachia-based tech- nologies for insect pest population con- trol. Adv Exp Med Biol. 627: 104–113. 29. Depm V, Mendes AM, Mauricio IL, Calado MM, Novo MT, Belo S (2016) Molecu- lar detection of Wolbachia pipientis in natural populations of mosquito vectors of Dirofilaria immitis from continental Portugal: first detection in Culex theileri. Med Vet Entomol. 30(3): 301–309. 30. Dyab AK, Galal LA, Mahmoud AE, Mokh- tar Y (2016) Finding Wolbachia in fi- larial larvae and culicidae mosquitoes in upper Egypt governorate. Korean J Par- asitol. 54(3): 265–272. 31. Zha X, Zhang W, Zhou C, Zhang L, Xiang Z, Xia Q (2014) Detection and charac- terization of Wolbachia infection in silk- worm. Genet Mol Biol. 37(3): 573–580. 32. Poorjavad N, Goldansaz SH, Machtelinckx T, Tirry L, Stouthamer R, van Leeuwen T (2012) Iranian Trichogramma: ITS2 DNA characterization and natural Wolbachia infection. BioControl. 57(3): 361–374. 33. Tijsse-Klasen E, Braks M, Scholte EJ, Sprong H (2011) Parasites of vectors-- Ixodiphagus hookeri and its Wolbachia symbionts in ticks in the Netherlands. Parasit Vectors. 4: 228. 34. Bereczki J, Rácz R, Varga Z, Tóth JP (2015) Controversial patterns of Wolbachia in- festation in the social parasitic Maculinea butterflies (Lepidoptera: Lycaenidae). Org Divers Evol. 15(3): 591–607. 35. Tabar MD, Altet L, Martinez V, Roura X (2013) Wolbachia, filariae and Leishma- nia coinfection in dogs from a Mediter- ranean area. J Small Anim Pract. 54(4): 174–178. 36. Simsek S, Ciftci AT (2016) Serological and molecular detection of Dirofilaria species in stray dogs and investigation of Wolbachia DNA by PCR in Turkey. J Arthropod Borne Dis. 10(4): 445–453. 37. Rossi MI, Aguiar-Alves F, Santos S, Paiva J, Bendas A, Fernandes O (2010) Detec- tion of Wolbachia DNA in blood from dogs infected with Dirofilaria immitis. Exp Parasitol. 126(2): 270–272. 38. Landum M, Ferreira CC, Calado M, Alho AM, Mauricio IL, Meireles JS (2014) Detection of Wolbachia in Dirofilaria infected dogs in Portugal. Vet Parasitol. 204(3–4): 407–410. 39. Morchón R, Carretón E, Grandi G, Gonzá- lez-Miguel J, Montoya-Alonso JA, Simón J Arthropod-Borne Dis, March 2019, 13(1): 83–93 M Khanmohammadi et al.: Molecular Detection and … 93 http://jad.tums.ac.ir Published Online: April 27, 2019 F (2012) 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. Vec- tor Borne Zoonotic Dis. 12(1): 17–20. 40. Enemark HL, Oksanen A, Chriél M, le Fèvre Harslund J, Woolsey ID, Al-Sabi MNS (2017) Detection and molecular characterization of the mosquito-borne filarial nematode Setaria tundra in Dan- ish roe deer (Capreolus capreolus). Int J Parasitol Parasites Wildl. 6(1): 16–21. 41. Comandatore F, Cordaux R, Bandi C, Blax- ter M, Darby A, Makepeace BL (2015) Supergroup C Wolbachia, mutualist sym- bionts of filarial nematodes, have a dis- tinct genome structure Biol Open. 5(12): 150099. 42. Taylor MJ, Voronin D, Johnston KL, Ford L (2013) Wolbachia filarial interactions. Cell Microbiol. 15(4): 520–526. 43. Armoo S, Doyle SR, Osei-Atweneboana MY, Grant WN (2017) Significant heter- ogeneity in Wolbachia copy number with- in and between populations of Onchocer- ca volvulus. Parasit Vectors. 10(1): 188. 44. Belanger DH, Perkins SL (2010) Wolbach- ia infection and mitochondrial diversity in the canine heartworm (Dirofilaria im- mitis). Mitochondrial DNA. 21(6): 227– 233. 45. Bazzocchi C, Jamnongluk W, O'Neill SL, Anderson TJ, Genchi C, Bandi C (2000) WSP gene sequences from the Wolbach- ia of filarial nematodes. Curr Microbi- ol. 41(2): 96–100. 46. Bandi C, Trees AJ, Brattig NW (2001) Wolbachia in filarial nematodes: evolu- tionary aspects and implications for the pathogenesis and treatment of filarial dis- eases. Vet Parasitol. 98(1–3): 215–238.