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Detecting and Identifying Wolbachia pipientis in Occult Dirofilariasis Using a High-resolution Melting Real-time PCR in Stray Dogs | ||
Iranian Journal of Veterinary Medicine | ||
مقاله 11، دوره 18، شماره 4، دی 2024، صفحه 579-588 اصل مقاله (3.67 M) | ||
نوع مقاله: Original Articles | ||
شناسه دیجیتال (DOI): 10.32598/ijvm.18.4.1005415 | ||
نویسندگان | ||
Fateme Manshori1؛ Fatemah Jalousian* 2؛ Seyed Hossein Hosseini1؛ Parviz Shayan3؛ Minoo Soltani4 | ||
1Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. | ||
2parasitology | ||
3Departmaent of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran | ||
4Rastegar Reference Laboratory, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. | ||
چکیده | ||
Background: Iran is one of the endemic areas of Dirofilaria immitis. The modified Knott test is the common diagnostic test based on detecting and identifying microfilaria in blood samples. Still, the results may be compromised by D. immitis occult infection (D. immitis infection without microfilariae). Wolbachia pipientis in a dog’s blood is an endosymbiont of D. immitis and an indicator of dirofilariasis. Objectives: This study aimed to investigate the occult infection of D. immitis in stray dogs in Guilan, Qazvin, and Mazandaran provinces, Iran, based on W. pipientis DNA tracking. Methods: Blood samples (n=138) were collected, and the presence of W. pipientis was evaluated by amplifying a partial sequence of the FtsZ gene (267 bp). Results: The results showed that 17.4% of the samples were positive by the modified Knott method, while 47.8% were positive by the molecular methods. The results of high-resolution melting (HRM) real-time PCR showed that the melting temperature of cox1 gene amplicons of D. immitis and Acanthocheilonema reconditum were obtained at 76.67±0.03 °C and 81.38±0.05 °C, respectively as well as it was achieved 80.30±0.04 °C for FtsZ gene of W. pipientis. The results of the present study showed that it is necessary to use molecular methods to diagnose dirofilariasis occult infection accurately. Conclusion: The HRM real-time PCR analysis is a simple post-PCR step that exploits the thermal characteristics of the amplicons to detect the occult infection of dirofilariasis based on W. pipientis DNA tracking and identification. | ||
کلیدواژهها | ||
Acanthocheilonema reconditum؛ Dirofilaria immitis؛ High-resolution melting (HRM) real-time PCR؛ Nematodes؛ Stray dogs | ||
اصل مقاله | ||
Introduction
The sample donated from the Iran Parasitology Museum was a thin male D. immitis 18 cm long, with a white tail with the typical spiral seen in Figure 1.
The measuring results of microfilaria using AxioVision® software, version 4.1 (Zeiss, Göttingen, Germany) illustrated that the length and width of D. immitis microfilariae occurred 295.13±14.9×5.8±0.43 µm and A. reconditum microfilariae was 228±12.1×5.8±0.43 µm, that computed using the digital camera (IDS uEye UI-2250SE USB 2.0 camera, Obersulm, Germany) under 40× magnification (Figure 2).
The results of the melting curve analysis of W. pipientis FtsZ gene amplicons shown in Figure 3 are the outcome of some samples assessed using HRM real-time PCR.
The modified Knott test has been used as one of the most common methods for diagnosing dirofilariasis in dogs. It is a recommended test for detecting microfilariae in peripheral blood or other body fluids as a concentrated method with relatively rapidity and cost-effectiveness. However, the challenge is still faced when the samples have a low number of microfilaremia and samples reveal an occult infection (Hoseini et al., 2020). Other assays, such as molecular methods, have been developed for dirofilariasis diagnosis and can be used for epidemiologic investigations in endemic areas; moreover, accurate recognition of dirofilariasis is demanded in occult infection (Mathison et al., 2019).
Acknowledgments
Albonico, F., Loiacono, M., Gioia, G., Genchi, C., Genchi, M., & Mortarino, M. (2014). Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis. Veterinary Parasitology, 200(1-2), 128-132. [DOI:10.1016/j.vetpar.2013.11.027] [PMID] Borhani Zarandi, M., Hoseini, S. H., Jalousion, F., Etebar, F., & Vojgani, M. (2017). Evaluate Toxocara canis excretory-secretory antigens in experimental allergic encephalomyelitis (EAE). Iranian Journal of Veterinary Medicine, 11(2), 175-182. [Link] David Ola-Fadunsin, S., Bisola Abdulrauf, A., Ganiyu, I., Hussain, K., Motunrayo Ambali, H., & Elelu, N. (2023). The intensityofinfectionandpublichealthperception of potentially zoonotic intestinal parasites of dogs in Kwara Central, Nigeria. Iranian Journal of Veterinary Medicine, 17(2), 119-128. [DOI:10.32598/ijvm.17.2.1005295] Hajijafari Anaraki, M., Sheikhi, N., Haghbin Nazarpak, H., & Nikbahkt Brujeni, G. (2022). Real time detection of different variant strains of infectious bronchitis virus in trachea, lung and kidney of infected broiler chickens. Iranian Journal of Veterinary Medicine, 16(1), 57-63. [DOI:10.22059/IJVM.2021.321594.1005165] Hoseini, M., Jalousian, F., Hoseini, S. H., & Gerami Sadeghian, A. (2020). A cross sectional study on dirofilaria immitis and acanthocheilonema reconditum in sheepdogs in a western region in Iran. Veterinary Research Forum, 11(2), 185-190. [PMID] Hosseini, S. H., Manshori-Ghaishghorshagh, F., Ramezani, M., Nayebzadeh, H., Ahoo, M. B., Eslamian, A., (2022). Canine microfilaraemia in some regions of Iran. Parasites and Vectors, 15(1), 90. [DOI:10.1186/s13071-022-05209-7] [PMID] [PMCID] Khanmohammadi, M., Akhlaghi, L., Razmjou, E., Falak, R., Zolfaghari Emameh, R., & Mokhtarian, K., et al. (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. [PMID] Landum, M., Ferreira, C. C., Calado, M., Alho, A. M., Maurício, I. L., & Meireles, J. S., et al. (2014). Detection of Wolbachia in Dirofilaria infected dogs in Portugal. Veterinary Parasitology, 204(3-4), 407-410. [DOI:10.1016/j.vetpar.2014.05.027] [PMID] Mathison, B. A., Couturier, M. R., & Pritt, B. S. (2019). Diagnostic identification and differentiation of microfilariae. Journal of Clinical Microbiology, 57(10), e00706-19. [DOI:10.1128/JCM.00706-19] [PMID] [PMCID] Molazem, M., Ramezani, A., Soroori, S., Jafari Giv, Z., Shokrpoor, S., & Geissbuehler, U. (2022). Feasiability of using evidence-based virtopsy to answer the possible clinical and postmortem Questions, in veterinary practice. Iranian Journal of Veterinary Medicine, 16(3), 311-337. [DOI:10.22059/IJVM.2021.315021.1005144] Ola-Fadunsin, S. D., Abdulrauf, A. B., Ganiyu, I. A., Hussain, K., Ambali, H. M., & Elelu, N. (2023). The intensity of infectionand public health perception of potentially zoonotic intestinal parasites of dogs in Kwara Central, Nigeria. Iranian Journal of Veterinary Medicine, 17(2), 119-128. [Link] Panarese, R., Iatta, R., Mendoza-Roldan, J. A., Szlosek, D., Braff, J., & Liu, J., et al. (2020). Comparison of diagnostic tools for the detection of dirofilaria immitis infection in dogs. Pathogens (Basel, Switzerland), 9(6), 499. [DOI:10.3390/pathogens9060499] [PMID] [PMCID] Razmaraii, N., Sadegh, E. S., Babaei, H., Paykari, H., Esmaeilnia, K., & Froghy, L. (2013). Molecular survey of Canine Microfilariae species in East-Azerbaijan Province of Iran. Archives Of Razi Institute, 68(2), 125-129. [Link] Rojas, A., Rojas, D., Montenegro, V. M., & Baneth, G. (2015). 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. Parasites & Vectors, 8, [DOI:10.1186/s13071-015-0783-8] [PMID] [PMCID] Turba, M. E., Zambon, E., Zannoni, A., Russo, S., & Gentilini, F.(2012). Detection of Wolbachia DNA in blood for diagnosing filaria-associated syndromes in cats. Journal of Clinical Microbiology, 50(8), 2624-2630. [DOI:10.1128/JCM.00528-12] [PMID] [PMCID] Wysmołek, M. E., Klockiewicz, M., Długosz, E., & Wiśniewski, M. (2022). Canine antibody response against Dirofilaria repens in natural occult and microfilaremic infections. Comparative Immunology, Microbiology and Infectious Diseases, 86, [DOI: 10.1016/j.cimid.2022.101818] [PMID] | ||
مراجع | ||
Albonico, F., Loiacono, M., Gioia, G., Genchi, C., Genchi, M., & Mortarino, M. (2014). Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis. Veterinary Parasitology, 200(1-2), 128-132. [DOI:10.1016/j.vetpar.2013.11.027] [PMID]
Borhani Zarandi, M., Hoseini, S. H., Jalousion, F., Etebar, F., & Vojgani, M. (2017). Evaluate Toxocara canis excretory-secretory antigens in experimental allergic encephalomyelitis (EAE). Iranian Journal of Veterinary Medicine, 11(2), 175-182. [Link]
David Ola-Fadunsin, S., Bisola Abdulrauf, A., Ganiyu, I., Hussain, K., Motunrayo Ambali, H., & Elelu, N. (2023). The intensityofinfectionandpublichealthperception of potentially zoonotic intestinal parasites of dogs in Kwara Central, Nigeria. Iranian Journal of Veterinary Medicine, 17(2), 119-128. [DOI:10.32598/ijvm.17.2.1005295]
Hajijafari Anaraki, M., Sheikhi, N., Haghbin Nazarpak, H., & Nikbahkt Brujeni, G. (2022). Real time detection of different variant strains of infectious bronchitis virus in trachea, lung and kidney of infected broiler chickens. Iranian Journal of Veterinary Medicine, 16(1), 57-63. [DOI:10.22059/IJVM.2021.321594.1005165]
Hoseini, M., Jalousian, F., Hoseini, S. H., & Gerami Sadeghian, A. (2020). A cross sectional study on dirofilaria immitis and acanthocheilonema reconditum in sheepdogs in a western region in Iran. Veterinary Research Forum, 11(2), 185-190. [PMID]
Hosseini, S. H., Manshori-Ghaishghorshagh, F., Ramezani, M., Nayebzadeh, H., Ahoo, M. B., Eslamian, A., (2022). Canine microfilaraemia in some regions of Iran. Parasites and Vectors, 15(1), 90. [DOI:10.1186/s13071-022-05209-7] [PMID] [PMCID]
Khanmohammadi, M., Akhlaghi, L., Razmjou, E., Falak, R., Zolfaghari Emameh, R., & Mokhtarian, K., et al. (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. [PMID]
Landum, M., Ferreira, C. C., Calado, M., Alho, A. M., Maurício, I. L., & Meireles, J. S., et al. (2014). Detection of Wolbachia in Dirofilaria infected dogs in Portugal. Veterinary Parasitology, 204(3-4), 407-410. [DOI:10.1016/j.vetpar.2014.05.027] [PMID]
Mathison, B. A., Couturier, M. R., & Pritt, B. S. (2019). Diagnostic identification and differentiation of microfilariae. Journal of Clinical Microbiology, 57(10), e00706-19. [DOI:10.1128/JCM.00706-19] [PMID] [PMCID]
Molazem, M., Ramezani, A., Soroori, S., Jafari Giv, Z., Shokrpoor, S., & Geissbuehler, U. (2022). Feasiability of using evidence-based virtopsy to answer the possible clinical and postmortem Questions, in veterinary practice. Iranian Journal of Veterinary Medicine, 16(3), 311-337. [DOI:10.22059/IJVM.2021.315021.1005144]
Ola-Fadunsin, S. D., Abdulrauf, A. B., Ganiyu, I. A., Hussain, K., Ambali, H. M., & Elelu, N. (2023). The intensity of infectionand public health perception of potentially zoonotic intestinal parasites of dogs in Kwara Central, Nigeria. Iranian Journal of Veterinary Medicine, 17(2), 119-128. [Link]
Panarese, R., Iatta, R., Mendoza-Roldan, J. A., Szlosek, D., Braff, J., & Liu, J., et al. (2020). Comparison of diagnostic tools for the detection of dirofilaria immitis infection in dogs. Pathogens (Basel, Switzerland), 9(6), 499. [DOI:10.3390/pathogens9060499] [PMID] [PMCID]
Razmaraii, N., Sadegh, E. S., Babaei, H., Paykari, H., Esmaeilnia, K., & Froghy, L. (2013). Molecular survey of Canine Microfilariae species in East-Azerbaijan Province of Iran. Archives Of Razi Institute, 68(2), 125-129. [Link]
Rojas, A., Rojas, D., Montenegro, V. M., & Baneth, G. (2015). 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. Parasites & Vectors, 8, [DOI:10.1186/s13071-015-0783-8] [PMID] [PMCID]
Turba, M. E., Zambon, E., Zannoni, A., Russo, S., & Gentilini, F.(2012). Detection of Wolbachia DNA in blood for diagnosing filaria-associated syndromes in cats. Journal of Clinical Microbiology, 50(8), 2624-2630. [DOI:10.1128/JCM.00528-12] [PMID] [PMCID]
Wysmołek, M. E., Klockiewicz, M., Długosz, E., & Wiśniewski, M. (2022). Canine antibody response against Dirofilaria repens in natural occult and microfilaremic infections. Comparative Immunology, Microbiology and Infectious Diseases, 86, [DOI: 10.1016/j.cimid.2022.101818] [PMID]
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