Macedonian Veterinary Review

Original Scientific Article

Immunohistochemical expression of GDNF, p53 and Ki67 with TUNEL assay in canine non-neoplastic esophageal nodules induced by Spirocerca lupi

Sara Amoorahim

Amir Amniattalab *

Mac Vet Rev 2025; 48 (1): i - xiii

10.2478/macvetrev-2025-0014

Received: 03 July 2024

Received in revised form: 06 January 2025

Accepted: 14 January 2025

Available Online First: 10 February 2025

Published on: 15 March 2025

Correspondence: Amir Amniattalab, amir.amniattalab@iau.ac.ir

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Abstract

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<0.05) 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<0.05) 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.

Keywords: Spirocerca lupi, non-neoplastic nodules, GDNF, apoptosis, cellular proliferation

References

1. 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. https://doi.org/10.17221/1866-VETMED

2. 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. https://doi.org/10.1111/j.1740-8261.2001.tb00914.x PMid:11327359

3. 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. https://doi.org/10.1017/S0022149X10000039 PMid:20132587

4. 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. https://doi.org/10.1111/j.1365-3024.2011.01316.x PMid:21770972
5. 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. https://doi.org/10.2460/javma.22.09.0401 PMid:36476412
6. 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. https://doi.org/10.1007/s00436-023-07825-3 PMid:37014474 PMCid:PMC10172281
7. Rojas, A., Dvir, E., Baneth, G. (2020). Insights on Spirocerca lupi, the carcinogenic dog nematode. Trends Parasitol. 36(1): 52-63. https://doi.org/10.1016/j.pt.2019.10.004 PMid:31734099
8. 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. https://doi.org/10.1186/s13027-023-00522-x PMid:37496079 PMCid:PMC10373346
9. 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. https://doi.org/10.3390/pathogens10020124 PMid:33530324 PMCid:PMC7911836
10. 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. https://doi.org/10.1016/j.vetpar.2010.08.032 PMid:20888695
11. 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. https://doi.org/10.1111/j.1365-2613.2008.00606.x PMid:19134055 PMCid:PMC2669607
12. Airaksinen, M.S., Saarma, M. (2002). The GDNF family: Signalling, biological functions and therapeutic value. Nat Rev Neurosci. 3(5): 383-394. https://doi.org/10.1038/nrn812 PMid:11988777
13. 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. https://doi.org/10.1016/j.jcpa.2020.12.002 PMid:33494907
14. Halliez, M.C.M., Buret, A.G. (2015). Gastrointestinal parasites and the neural control of gut functions. Front Cell Neurosci. 9, 452. https://doi.org/10.3389/fncel.2015.00452 PMid:26635531 PMCid:PMC4658430
15. 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. https://doi.org/10.1590/S1984-29612016058 PMid:27683842
16. 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. https://doi.org/10.1007/s004320000183 PMid:11315258
17. 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. https://doi.org/10.2460/javma.228.7.1053 PMid:16579784
18. 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. https://doi.org/10.1034/j.1600-0463.2003.t01-1-1110208.x PMid:12752223
19. 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. https://doi.org/10.3748/wjg.v8.i2.200 PMid:11925591 PMCid:PMC4658350
20. Mirzayans, R., Murray, D. (2020). Do TUNEL and other apoptosis assays detect cell death in preclinical studies? Int J Mol Sci. 21(23): 9090. https://doi.org/10.3390/ijms21239090 PMid:33260475 PMCid:PMC7730366
21. 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. https://doi.org/10.1038/s41598-019-49344-1 PMid:31506501 PMCid:PMC6736856
22. 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. https://doi.org/10.1017/S0022149X01000555 PMid:11818054
23. 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. https://doi.org/10.1016/j.parint.2012.04.002 PMid:22562003
24. 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. https://doi.org/10.1177/0300985818800026 PMid:30278838
25. 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.
26. 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.
27. 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. https://doi.org/10.1016/j.vetpar.2008.04.006 PMid:18534758
28. 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. https://doi.org/10.7589/JWD-D-20-00162 PMid:34788835
29. 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.
30. 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. https://doi.org/10.1111/j.1439-0264.1998.tb00189.x PMid:9741148
31. 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. https://doi.org/10.1016/j.pathol.2023.05.009 PMid:37544878
32. 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. https://doi.org/10.3892/ol.2013.1226 PMid:23759874 PMCid:PMC3678859

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© 2025 Amoorahim S. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Conflict of Interest Statement

The authors declared that they have no potential lict of interest with respect to the authorship and/or publication of this article.

Citation Information

Macedonian Veterinary Review. Volume 48, Issue 1, Pages i-xiii, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2025-0014