Original Scientific Article Spirocercosis caused by Spirocerca lupi has a global distribution and mainly infects dogs although other carnivores may be affected as well. The research aimed to ascertain the proportion of cellular proliferation/apoptosis within the parasiteinduced nodules and to categorize the inflammatory cells by CD3 (for T lymphocytes), CD20 (for B lymphocytes), and CD68 (for macrophages) markers. The study included 152 Iranian mixed-breed dogs (87 males and 65 females) examined for esophageal spirocercosis. The dogs underwent hematological investigations and Telemann concentration stool exams to detect the parasites’ eggs. The normal and nodular esophagi underwent TUNEL assay, histopathology, and immunohistochemical staining for GDNF, p53, Ki67, CD3, CD20, and CD68. According to our findings, 31 out of 152 dogs (20.39%) were diagnosed with spirocercosis based on clinical examinations and stool exams, while 20 out of 152 dogs (13.16%) had nodular esophagi in necropsy. Normal and nodular esophageal tissue samples (n=64) were collected from necropsied dogs. Histopathology confirmed non-neoplastic parasitic nodular lesions. Immunohistochemically, increased GDNF, Ki67, CD3, CD20, and CD68 expression was significant (p<) in nodular tissue compared to normal tissue. However, the expression of p53 was not significant (p>0.05). Given the results of Ki67 expression and TUNEL assay, the rate of proliferation (6-8 times)/apoptosis (2-3 times) significantly increased (p<) in the infected tissue compared to normal tissue. The results indicated that the GDNF as a neurotrophic growth factor may play an important role in the pathogenesis of nodular spirocercosis in dogs. Meanwhile, a high proliferation/apoptosis rate in the parasitic nodular compared to normal esophagi may stimulate inducing neoplastic transformation in normal esophageal tissue. https://macvetrev.mk/LoadArticlePdf/361 2025-02-10 i xiii https://doi.org/10.2478/macvetrev-2025-0014 Spirocerca lupi non-neoplastic nodules GDNF apoptosis cellular proliferation Sara Amoorahim false 1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran AUTHOR Amir Amniattalab amir.amniattalab@iau.ac.ir false 2 Department of Pathology, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran LEAD_AUTHOR Oryan, A., Sadjjadi, S.M., Mehrabani, D., Kargar, M. (2008). Spirocercosis and its complications in stray dogs in Shiraz, southern Iran. Vet Med-Czech. 53(11): 617-624. 1 https://doi.org/10.17221/1866-VETMED Dvir, E., Kirberger, R.M., Malleczek, D. (2001). Radiographic and computed tomographic changes and clinical presentation of spirocercosis in the dog. Vet Radiol Ultrasound. 42(2): 119-129. PMid:11327359 2 https://doi.org/10.1111/j.1740-8261.2001.tb00914.x Mukaratirwa, S., Pillay, E., Munsammy, K. (2010). Experimental infection of selected arthropods with spirurid nematodes Spirocerca lupi Railliet & Henry, 1911 and Gongylonema ingluvicola Molin, 1857. J Helminthol. 84(4): 369-374. PMid:20132587 3 https://doi.org/10.1017/S0022149X10000039 Dvir, E., Schoeman, J.P., Clift, S.J., McNeilly, T.N., Mellanby, R.J. (2011). Immunohistochemical characterization of lymphocyte and myeloid cell infiltrates in spirocercosis-induced oesophageal nodules. Parasite Immunol. 33(10): 545-553. PMid:21770972 4 https://doi.org/10.1111/j.1365-3024.2011.01316.x Asiag, N., Chai, O., Yodovner, S., Ruggeri, M., Rapaport, K., Baneth, G., Nachum-Biala, Y., et al. (2022). Evaluation of a treatment protocol in dogs with intraspinal spirocercosis. J Am Vet Med Assoc. 261(3): 384-390. PMid:36476412 5 https://doi.org/10.2460/javma.22.09.0401 Di Cataldo, S., Cevidanes, A., Sepúlveda-García, P., Alvarado-Rybak, M., Lia, R.P., Otranto, D., Terio, K., et al. (2023). Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile. Parasitol Res. 122(6): 1261-1269. PMid:37014474 PMCid:PMC10172281 6 https://doi.org/10.1007/s00436-023-07825-3 Rojas, A., Dvir, E., Baneth, G. (2020). Insights on Spirocerca lupi, the carcinogenic dog nematode. Trends Parasitol. 36(1): 52-63. PMid:31734099 7 https://doi.org/10.1016/j.pt.2019.10.004 Fonti, N., Parisi, F., Mancianti, F., Freer, G., Poli, A. (2023). Cancerogenic parasites in veterinary medicine: a narrative literature review. Infect Agent Cancer. 18(1): 45. PMid:37496079 PMCid:PMC10373346 8 https://doi.org/10.1186/s13027-023-00522-x Porras-Silesky, C., Mejías-Alpízar, M.J., Mora, J., Baneth, G., Rojas, A. (2021). Spirocerca lupi proteomics and its role in cancer development: An overview of spirocercosis-induced sarcomas and revision of helminth-induced carcinomas. Pathogens 10(2): 124. PMid:33530324 PMCid:PMC7911836 9 https://doi.org/10.3390/pathogens10020124 Dvir, E., Clift, S.J. (2010). Evaluation of selected growth factor expression in canine spirocercosis (Spirocerca lupi)-associated non-neoplastic nodules and sarcomas. Vet Parasitol. 174(3-4): 257-266. PMid:20888695 10 https://doi.org/10.1016/j.vetpar.2010.08.032 Starke-Buzetti, W.A., Oaks, J.A. (2008). Increased glial-derived neurotrophic factor in the small intestine of rats infected with the tapeworm, Hymenolepis diminuta. Int J Exp Pathol. 89(6): 458-465. PMid:19134055 PMCid:PMC2669607 11 https://doi.org/10.1111/j.1365-2613.2008.00606.x Airaksinen, M.S., Saarma, M. (2002). The GDNF family: Signalling, biological functions and therapeutic value. Nat Rev Neurosci. 3(5): 383-394. PMid:11988777 12 https://doi.org/10.1038/nrn812 Khodamoradi, P., Amniattalab, A., Alizadeh, S. (2021). Overexpression of GDNF and FGF-1 in canine benign prostatic hyperplasia: evidence for a pathogenetic role of neural growth factor. J Comp Pathol. 182, 43-53. PMid:33494907 13 https://doi.org/10.1016/j.jcpa.2020.12.002 Halliez, M.C.M., Buret, A.G. (2015). Gastrointestinal parasites and the neural control of gut functions. Front Cell Neurosci. 9, 452. PMid:26635531 PMCid:PMC4658430 14 https://doi.org/10.3389/fncel.2015.00452 El-Aal, A.A.A., El-Gebaly, N.S.M., Al-Antably, A.S., Hassan, M.A., El-Dardiry, M.A. (2016). Post-immunization immunohistochemical expression of Caspase 3 and p53 apoptotic markers in experimental hydatidosis. Rev Bras Parasitol Vet. 25(3): 333-340. PMid:27683842 15 https://doi.org/10.1590/S1984-29612016058 Zhang, W., Rashid, A., Wu, H., Xiao-Chun, X. (2001). Differential expression of retinoic acid receptors in normal and malignant esophageal tissues. J Cancer Res Clin Oncol. 127(4): 237-242. PMid:11315258 16 https://doi.org/10.1007/s004320000183 Ettinger, S.N., Scase, T.J., Oberthaler, K.T., Craft, D.M., McKnight, J.A., Leibman, N.F., Charney, S.C., Bergman, P.J. (2006). Association of argyrophilic nucleolar organizing regions, Ki-67, and proliferating cell nuclear antigen scores with histologic grade and survival in dogs with soft tissue sarcomas: 60 Cases (1996-2002). J Am Vet Med Assoc. 228(7): 1053-1062. PMid:16579784 17 https://doi.org/10.2460/javma.228.7.1053 Zacchetti, A., Van Garderen, E., Teske, E., Nederbragt, H., Dierendonck, J.H., Rutteman, G.R. (2003). Validation of the use of proliferation markers in canine neoplastic and non-neoplastic tissues: Comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry. APMIS 111(3): 430-438. PMid:12752223 18 https://doi.org/10.1034/j.1600-0463.2003.t01-1-1110208.x Xu, M., Jin, Y.L., Fu, J., Huang, H., Chen, S.Z., Qu, P., Tian, H.M., et al. (2002). The abnormal expression of retinoic acid receptor-β, p53 and Ki67 protein in normal, premalignant and malignant esophageal tissues. World J Gastroenterol. 8(2): 200-202. PMid:11925591 PMCid:PMC4658350 19 https://doi.org/10.3748/wjg.v8.i2.200 Mirzayans, R., Murray, D. (2020). Do TUNEL and other apoptosis assays detect cell death in preclinical studies? Int J Mol Sci. 21(23): 9090. PMid:33260475 PMCid:PMC7730366 20 https://doi.org/10.3390/ijms21239090 De Souza, T.L., Da Silva, A.V.A., Pereira, O.R., Figueiredo, F.B., Mendes Junior, A.A.V., Menezes, R.C., Mendes-da-Cruz, D.A., et al. (2019). Pro-cellular exhaustion markers are associated with splenic microarchitecture disorganization and parasite load in dogs with visceral leishmaniasis. Sci Rep. 9(1): 12962. PMid:31506501 PMCid:PMC6736856 21 https://doi.org/10.1038/s41598-019-49344-1 Mylonakis, M.E., Koutinas, A.F., Liapi, M.V., Saridomichelakis, M.N., Rallis, T.S. (2001). A comparison of the prevalence of Spirocerca lupi in three groups of dogs with different life and hunting styles. J Helminthol. 75(4): 359-361. PMid:11818054 22 https://doi.org/10.1017/S0022149X01000555 Saini, P., Gayen, P., Nayak, A., Kumar, D., Mukherjee, N., Pal, B.C., Sinha Babu, S.P. (2012). Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: Molecular and biochemical approaches. Parasitol Int. 61(4): 520-531. PMid:22562003 23 https://doi.org/10.1016/j.parint.2012.04.002 Janke, L.J., Ward, J.M., Vogel, P. (2019). Classification, scoring, and quantification of cell death in tissue sections. Vet Pathol. 56(1): 33-38. PMid:30278838 24 https://doi.org/10.1177/0300985818800026 Alatzas, D.G., Brellou, G., Psychas, V., Papadopoulou, P., Mylonakis, M., Soubasis, N., Rallis, T. (2014). Spirocerca lupi-associated pyothorax in two dogs. Hell J Companion Anim Med. 3(1): 60-69. 25 Couto, C.G. (2009). Hematology. In: Nelson, R.W. Nelson, C.G. Couto. (Eds.), Small Animal Internal Medicine. (pp. 1209-1278), 4th ed. Mosby, Inc. 26 Dvir, E., Kirberger, R.M., Mukorera, V., Van der Merwe, L.L., Clift, S.J. (2008). Clinical differentiation between dogs with benign and malignant spirocercosis. Vet Parasitol. 155(1-2): 80-88. PMid:18534758 27 https://doi.org/10.1016/j.vetpar.2008.04.006 Martín-Pérez, M., Gómez-Gordo, L., Galapero, J., Pérez-Martín, J.E., Bravo-Barriga, D., Calero-Bernal, R., Frontera, E. (2022). Characterization of lesions induced by Spirocerca vulpis (Spiruridae: Spirocercidae) in Red Foxes (Vulpes vulpes). J Wildl Dis. 58(1): 137-147. PMid:34788835 28 https://doi.org/10.7589/JWD-D-20-00162 Azarabad, H., Gharagozlou, M.J., Nowrouzian, I., Seyedjavad, M.R. (2011). P53 and Ki67 protein expression in ocular squamous cell carcinomas of dairy cattle. Int J Vet Res. 5(4): 226-231. 29 Laprie, C., Abadie, J., Amardeilh, M.F., Raymond, I., Delverdier, M. (1998). Detection of the Ki-67 proliferation associated nuclear epitope in normal canine tissues using the monoclonal antibody MIB-1. Anat Histol Embryol. 27(4): 251-256. PMid:9741148 30 https://doi.org/10.1111/j.1439-0264.1998.tb00189.x Jeong, J.S., Cho, K.J., Lee, H.J., Roh, J., Lee, Y.S., Song, J.S. (2023). Predictive modelling for the diagnosis of oral and laryngeal premalignant and malignant lesions using p53 and Ki-67 expression. Pathology. 55(7): 945-957. PMid:37544878 31 https://doi.org/10.1016/j.pathol.2023.05.009 Campos, M., De Campos, S.G.P., Ribeiro, G.G., Eguchi, F.C., Da Silva, S.R.M., De Oliveira, C.Z., Da Costa, A.M., et al. (2013). Ki-67 and CD100 immunohistochemical expression is associated with local recurrence and poor prognosis in soft tissue sarcomas, respectively. Oncol Lett. 5(5): 1527-1535. PMid:23759874 PMCid:PMC3678859 32 https://doi.org/10.3892/ol.2013.1226