Research Article
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Year 2023, Volume: 8 Issue: 3, 216 - 225, 15.12.2023

Emin Karakurt Semistan Kızıltepe Ayfer Yıldız

https://doi.org/10.31797/vetbio.1364749

Abstract

References

  • Asmare, K., Sibhat, B., Haile, A., Sheferaw, D., Aragaw, K., Abera, M., Abebe, R., & Wieland, B. (2018). Lungworm infection in small ruminants in Ethiopia: Systematic review and meta-analysis. Veterinary Parasitology,
  • Regional Studies and Reports, 14, 63–70. https://doi.org/10.1016/j.vprsr.2018.08.003
  • Attia, M. M., Saad, M. F., & Khalf, M. A. (2022). Milk tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interferon (IFN-γ) and oxidative stress markers as new indicators for fascioliasis in dairy water buffaloes. Microbial Pathogenesis, 169, 105672. https://doi.org/10.1016/j.micpath.2022.105672
  • Beytut, B., Akça, A., & Gökçe, H. İ. (2011). Pathological and immunohistochemical evaluation of the effects of interferon gamma (IFN-γ) and aminoguanidine in rats experimentally infected with Fasciola hepatica. Turkish Journal of Veterinary & Animal Sciences, 35(4), 243-253. https://doi.org/10.3906/vet-1004-302
  • Beytut, E., Otlu, S., & Sözmen, M. (2002). Kars bölgesi koyunlarında gözlenen pnömoniler üzerine patolojik ve etiyolojik incelemeler. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 8(1), 31-39.
  • Bouljihad, M., Berrag, B., & Leipold, H. W. (1995). Gross and light-microscopic features of ovine pulmonary hydatidosis and verminous pneumonias in Morocco. Zentralblatt fur Veterinarmedizin. Reihe B. Journal of Veterinary Medicine. Series B, 42(9), 513–521. https://doi.org/10.1111/j.1439-0450.1995.tb00743.x
  • Can, V., Özdal, N., & Oğuz, B. (2018). Prevalence of lungworms with macroscopically verminous pneumonia and in sheep with respiratory syndrome. Van Veterinary Journal, 29(3), 153-157.
  • de Macedo, L. O., Lima, T. A. R. F., Verocai, G. G., Alves, L. C., de Carvalho, G. A., & Ramos, R. A. N. (2021). Lungworms in ruminants from Brazil: A retrospective epidemiological study over four decades. Veterinary Parasitology, Regional Studies and Reports, 26, 100645. https://doi.org/10.1016/j.vprsr.2021.100645
  • Fesseha, H., & Mathewos, M. (2021). Prevalence and risk factors of bovine and ovine lungworm infection at Durame District, Southern Ethiopia. Journal of Parasitology Research, 2021, 6637718. https://doi.org/10.1155/2021/6637718
  • Fleury, A., Alaez, C., Dessein, A., Rosetti, M., Saenz, B., Hernández, M., Bobes, R. J., Ramírez-Aquino, R., Sciutto, E., Gorodezky, C., & Fragoso, G. (2018). No association of IL2, IL4, IL6, TNF, and IFNG gene polymorphisms was found with Taenia solium human infection or neurocysticercosis severity in a family-based study. Human Immunology, 79(7), 578–582. https://doi.org/10.1016/j.humimm.2018.04.011
  • Gulbahar, M. Y., Davis, W. C., Yarim, M., Guvenc, T., Umur, S., Kabak, Y. B., Karayigit, M. O., & Beyhan, Y. E. (2009). Characterization of local immune response against lungworms in naturally infected sheep. Veterinary Parasitology, 160(3-4), 272–278. https://doi.org/10.1016/j.vetpar.2008.11.012
  • Hanks, J. E., Campbell, A. J. D., & Larsen, J. W. A. (2021). Severity and prevalence of small lungworm infection on three South Australian farms and associations with sheep carcass characteristics. Veterinary Parasitology, 296, 109503. https://doi.org/10.1016/j.vetpar.2021.109503
  • Hanks, J. E., Larsen, J., & Campbell, A. (2022). Factors associated with small lungworm infections in heavily infected sheep in southeast South Australia. Australian Veterinary Journal, 100(1-2), 20–28. https://doi.org/10.1111/avj.13123
  • Hashemnia, M., Chalechale, A., & Malmir, E. (2019). Pulmonary lesions in slaughtered sheep in Western Iran: Gross and histopathological findings. Veterinaria Italiana, 55(1), 47–56. https://doi.org/10.12834/VetIt.785.3795.3
  • Holmgren, S., Hagberg Gustavsson, M., Lundén, A., & Wattrang, E. (2014). Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves. Parasite Immunology, 36(2), 78–86. https://doi.org/10.1111/pim.12083
  • Jabbar, A., Mohandas, N., Jex, A. R., & Gasser, R. B. (2013). The mitochondrial genome of Protostrongylus rufescens - implications for population and systematic studies. Parasites & Vectors, 6(1), 263. https://doi.org/10.1186/1756-3305-6-263
  • Johnson, D. R., Sales, J., & Matthews, J. B. (2005). Local cytokine responses in Dictyocaulus viviparus infection. Veterinary Parasitology, 128(3-4), 309–318. https://doi.org/10.1016/j.vetpar.2004.12.001
  • Kak, G., Raza, M., & Tiwari, B. K. (2018). Interferon-gamma (IFN-γ): Exploring its implications in infectious diseases. Biomolecular Concepts, 9(1), 64–79. https://doi.org/10.1515/bmc-2018-0007
  • Karakurt, E., & Dağ, S. (2020). Protective effect of silymarin on diethylnitrosamine induced hepatocellular carcinoma model in wistar rats; cancer patterns and cytological variants. Atatürk Üniv Vet Bil Derg, 15(1), 43-50. https://doi.org/10.17094/ataunivbd.680945
  • Karakurt, E., Nuhoğlu, H., Dağ, S., Çelebi, Ö., Büyük, F., Beytut, E., Yıldız, A., Kuru, M., & Akça, D. (2023). Immunohistochemical investigation of TNF-α and IFN-γ expressions in sheep fetuses with Brucellosis. Pakistan Veterinary Journal, 43(1), 85-90. http://dx.doi.org/10.29261/pakvetj/2022.088
  • Kıran, M. M., Berkin, Ş., Kaya, O., & Dinçer, Z. (1993). Konya bölgesi koyun pnömonilerinde patolojik ve etiyolojik araştırmalar. Selçuk Üniversitesi Veteriner Fakültesi Dergisi, 9(1), 3-9.
  • Kırcalı Sevimli, F., Kozan, E., & Doğan, N. (2011). Efficacy of eprinomectin pour-on treatment in sheep naturally infected with Dictyocaulus filaria and Cystocaulus ocreatus. Journal of Helminthology, 85(4), 472–475. https://doi.org/10.1017/S0022149X10000854
  • Kidane, F. A., Mitra, T., Wernsdorf, P., Hess, M., & Liebhart, D. (2018). Allocation of interferon gamma mRNA positive cells in caecum hallmarks a protective trait against Histomonosis. Frontiers in Immunology, 9, 1164. https://doi.org/10.3389/fimmu.2018.01164
  • Kim, W. H., Chaudhari, A. A., & Lillehoj, H. S. (2019). Involvement of T cell immunity in avian coccidiosis. Frontiers in Immunology, 10, 2732. https://doi.org/10.3389/fimmu.2019.02732
  • Mangiola, S., Young, N. D., Sternberg, P. W., Strube, C., Korhonen, P. K., Mitreva, M., Scheerlinck, J. P., Hofmann, A., Jex, A. R., & Gasser, R. B. (2014). Analysis of the transcriptome of adult Dictyocaulus filaria and comparison with Dictyocaulus viviparus, with a focus on molecules involved in host-parasite interactions. International Journal for Parasitology, 44(3-4), 251–261. https://doi.org/10.1016/j.ijpara.2013.12.003
  • Manirarora, J. N., Walker, K. E., Patil, V., Renukaradhya, G. J., LaBresh, J., Sullivan, Y., Francis, O., & Lunney, J. K. (2022). Development and characterization of new monoclonal antibodies against porcine interleukin-17A and interferon-gamma. Frontiers in Immunology, 13, 786396. https://doi.org/10.3389/fimmu.2022.786396
  • Papadopoulos E, Sotiraki S, Himonas C, & Fthenakis GC. (2004). Treatment of small lungworm infestation in sheep by using moxidectin. Veterinary Parasitology, 121(3-4), 329–336. https://doi.org/10.1016/j.vetpar.2004.02.026
  • Panayotova-Pencheva, M. S., & Alexandrov, M. T. (2010). Some pathological features of lungs from domestic and wild ruminants with single and mixed protostrongylid infections. Veterinary Medicine International, 2010, 741062. https://doi.org/10.4061/2010/741062
  • Panuska, C. (2006). Lungworms of ruminants. Veterinary Clinics of North America: Food Animal Practice, 22(3), 583–593. https://doi.org/10.1016/j.cvfa.2006.06.002
  • Ram, P. K., Singh, S. K., Kumari, P., Srivastava, M., Sudan, V., Pandey, R. P., & Garg, S. K. (2020). Role of cytokines in the clinical manifestation of exophthalmia in newborn calves with tropical theileriosis. Parasite Immunology, 42(10), e12761.
  • Rehbein, S., Knaus, M., Li, J., Antretter, A., Mayr, S., Visser, M., & Hamel, D. (2022). Treatment of natural Protostrongylus rufescens lungworm infection in sheep with eprinomectin 5 mg/mL topical solution. Veterinary Parasitology, 301, 109639. https://doi.org/10.1016/j.vetpar.2021.109639
  • Rehbein, S., & Visser, M. (2002). Efficacy of ivermectin delivered via a controlled-release capsule against small lungworms (Protostrongylidae) in sheep. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health, 49(7), 313–316. https://doi.org/10.1046/j.1439-0450.2002.00576.x
  • Sağlam, Y. S., Bozoğlu, H., & Baş, A. T. (1998). Erzurum ve çevresinde kuzu enzootik pnömonileri üzerinde bakteriyolojik ve patolojik incelemeler. Etlik Veteriner Mikrobiyoloji Dergisi, 10(1), 1-17.
  • Samar, A. E., Bahaa, E. D., & Shereen, F. M. (2017). Parasitological, pathological and immunological effects of Nigella sativa oil in experimentally Toxocara canis infected mice. Tropical Biomedicine, 34(4), 781–794.
  • Sasai, M., & Yamamoto, M. (2019). Innate, adaptive, and cell-autonomous immunity against Toxoplasma gondii infection. Experimental & Molecular Medicine, 51(12), 1–10. https://doi.org/10.1038/s12276-019-0353-9
  • Ülgen, M., Sönmez, G., & Aydın, F. (1997). Kuzu pnömonileri üzerinde mikrobiyolojik ve histopatolojik incelemeler. Etlik Veteriner Mikrobiyoloji Dergisi, 9(2), 55-70.
  • Zafari, S., Mohtasebi, S., Sazmand, A., Bahari, A., Sargison, N. D., & Verocai, G. G. (2022). The prevalence and control of lungworms of pastoral ruminants in Iran. Pathogens, 11(12), 1392. https://doi.org/10.3390/pathogens11121392

Immunohistochemical evaluation of IFN-γ levels in sheep verminous pneumonia

Year 2023, Volume: 8 Issue: 3, 216 - 225, 15.12.2023

Emin Karakurt Semistan Kızıltepe Ayfer Yıldız

https://doi.org/10.31797/vetbio.1364749

Abstract

Lungworms, a group of parasitic nematodes, are recognized as one of the major and most common parasitic pneumonia agents in ruminants worldwide. In this study, the expressions of interferon gamma (IFN-γ), which is an important pro-inflammatory cytokine, were evaluated by immunohistochemical methods in order to evaluate the immune response against parasitic agents in sheep naturally infected with different types of lungworms. The material for this study consisted of lung tissue samples obtained from 40 dead sheep brought for routine histopathological diagnosis to the Department of Pathology. In order to reveal the histopathological changes in the tissues, Hematoxylin and Eosin (H&E) staining was applied to the sections. Lung tissues were stained with IFN-γ commercial antibody using the Avidin-Biotin Peroxidase Technique (ABC) following the procedures of the manufacturer. Subpleural multifocal nodules of several mm in diameter were detected in the dorsal regions of the lung, especially in the caudal lobes. In the histopathological examination of the lungs, it was observed that the alveoli, bronchi, and bronchiole lumens were filled with adult forms, larvae, and eggs of the parasitic agents. Compared to the control group, the expressions of IFN-γ were significantly increased in the verminous pneumonia group. Overall, the study suggests that the Th1 response, as represented by increased IFN-γ expression, appears to play an active role in the immunity developed against lungworms in ruminants.

Keywords

Cytokine IFN-γ lungworms sheep verminous pneumonia

References

  • Asmare, K., Sibhat, B., Haile, A., Sheferaw, D., Aragaw, K., Abera, M., Abebe, R., & Wieland, B. (2018). Lungworm infection in small ruminants in Ethiopia: Systematic review and meta-analysis. Veterinary Parasitology,
  • Regional Studies and Reports, 14, 63–70. https://doi.org/10.1016/j.vprsr.2018.08.003
  • Attia, M. M., Saad, M. F., & Khalf, M. A. (2022). Milk tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interferon (IFN-γ) and oxidative stress markers as new indicators for fascioliasis in dairy water buffaloes. Microbial Pathogenesis, 169, 105672. https://doi.org/10.1016/j.micpath.2022.105672
  • Beytut, B., Akça, A., & Gökçe, H. İ. (2011). Pathological and immunohistochemical evaluation of the effects of interferon gamma (IFN-γ) and aminoguanidine in rats experimentally infected with Fasciola hepatica. Turkish Journal of Veterinary & Animal Sciences, 35(4), 243-253. https://doi.org/10.3906/vet-1004-302
  • Beytut, E., Otlu, S., & Sözmen, M. (2002). Kars bölgesi koyunlarında gözlenen pnömoniler üzerine patolojik ve etiyolojik incelemeler. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 8(1), 31-39.
  • Bouljihad, M., Berrag, B., & Leipold, H. W. (1995). Gross and light-microscopic features of ovine pulmonary hydatidosis and verminous pneumonias in Morocco. Zentralblatt fur Veterinarmedizin. Reihe B. Journal of Veterinary Medicine. Series B, 42(9), 513–521. https://doi.org/10.1111/j.1439-0450.1995.tb00743.x
  • Can, V., Özdal, N., & Oğuz, B. (2018). Prevalence of lungworms with macroscopically verminous pneumonia and in sheep with respiratory syndrome. Van Veterinary Journal, 29(3), 153-157.
  • de Macedo, L. O., Lima, T. A. R. F., Verocai, G. G., Alves, L. C., de Carvalho, G. A., & Ramos, R. A. N. (2021). Lungworms in ruminants from Brazil: A retrospective epidemiological study over four decades. Veterinary Parasitology, Regional Studies and Reports, 26, 100645. https://doi.org/10.1016/j.vprsr.2021.100645
  • Fesseha, H., & Mathewos, M. (2021). Prevalence and risk factors of bovine and ovine lungworm infection at Durame District, Southern Ethiopia. Journal of Parasitology Research, 2021, 6637718. https://doi.org/10.1155/2021/6637718
  • Fleury, A., Alaez, C., Dessein, A., Rosetti, M., Saenz, B., Hernández, M., Bobes, R. J., Ramírez-Aquino, R., Sciutto, E., Gorodezky, C., & Fragoso, G. (2018). No association of IL2, IL4, IL6, TNF, and IFNG gene polymorphisms was found with Taenia solium human infection or neurocysticercosis severity in a family-based study. Human Immunology, 79(7), 578–582. https://doi.org/10.1016/j.humimm.2018.04.011
  • Gulbahar, M. Y., Davis, W. C., Yarim, M., Guvenc, T., Umur, S., Kabak, Y. B., Karayigit, M. O., & Beyhan, Y. E. (2009). Characterization of local immune response against lungworms in naturally infected sheep. Veterinary Parasitology, 160(3-4), 272–278. https://doi.org/10.1016/j.vetpar.2008.11.012
  • Hanks, J. E., Campbell, A. J. D., & Larsen, J. W. A. (2021). Severity and prevalence of small lungworm infection on three South Australian farms and associations with sheep carcass characteristics. Veterinary Parasitology, 296, 109503. https://doi.org/10.1016/j.vetpar.2021.109503
  • Hanks, J. E., Larsen, J., & Campbell, A. (2022). Factors associated with small lungworm infections in heavily infected sheep in southeast South Australia. Australian Veterinary Journal, 100(1-2), 20–28. https://doi.org/10.1111/avj.13123
  • Hashemnia, M., Chalechale, A., & Malmir, E. (2019). Pulmonary lesions in slaughtered sheep in Western Iran: Gross and histopathological findings. Veterinaria Italiana, 55(1), 47–56. https://doi.org/10.12834/VetIt.785.3795.3
  • Holmgren, S., Hagberg Gustavsson, M., Lundén, A., & Wattrang, E. (2014). Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves. Parasite Immunology, 36(2), 78–86. https://doi.org/10.1111/pim.12083
  • Jabbar, A., Mohandas, N., Jex, A. R., & Gasser, R. B. (2013). The mitochondrial genome of Protostrongylus rufescens - implications for population and systematic studies. Parasites & Vectors, 6(1), 263. https://doi.org/10.1186/1756-3305-6-263
  • Johnson, D. R., Sales, J., & Matthews, J. B. (2005). Local cytokine responses in Dictyocaulus viviparus infection. Veterinary Parasitology, 128(3-4), 309–318. https://doi.org/10.1016/j.vetpar.2004.12.001
  • Kak, G., Raza, M., & Tiwari, B. K. (2018). Interferon-gamma (IFN-γ): Exploring its implications in infectious diseases. Biomolecular Concepts, 9(1), 64–79. https://doi.org/10.1515/bmc-2018-0007
  • Karakurt, E., & Dağ, S. (2020). Protective effect of silymarin on diethylnitrosamine induced hepatocellular carcinoma model in wistar rats; cancer patterns and cytological variants. Atatürk Üniv Vet Bil Derg, 15(1), 43-50. https://doi.org/10.17094/ataunivbd.680945
  • Karakurt, E., Nuhoğlu, H., Dağ, S., Çelebi, Ö., Büyük, F., Beytut, E., Yıldız, A., Kuru, M., & Akça, D. (2023). Immunohistochemical investigation of TNF-α and IFN-γ expressions in sheep fetuses with Brucellosis. Pakistan Veterinary Journal, 43(1), 85-90. http://dx.doi.org/10.29261/pakvetj/2022.088
  • Kıran, M. M., Berkin, Ş., Kaya, O., & Dinçer, Z. (1993). Konya bölgesi koyun pnömonilerinde patolojik ve etiyolojik araştırmalar. Selçuk Üniversitesi Veteriner Fakültesi Dergisi, 9(1), 3-9.
  • Kırcalı Sevimli, F., Kozan, E., & Doğan, N. (2011). Efficacy of eprinomectin pour-on treatment in sheep naturally infected with Dictyocaulus filaria and Cystocaulus ocreatus. Journal of Helminthology, 85(4), 472–475. https://doi.org/10.1017/S0022149X10000854
  • Kidane, F. A., Mitra, T., Wernsdorf, P., Hess, M., & Liebhart, D. (2018). Allocation of interferon gamma mRNA positive cells in caecum hallmarks a protective trait against Histomonosis. Frontiers in Immunology, 9, 1164. https://doi.org/10.3389/fimmu.2018.01164
  • Kim, W. H., Chaudhari, A. A., & Lillehoj, H. S. (2019). Involvement of T cell immunity in avian coccidiosis. Frontiers in Immunology, 10, 2732. https://doi.org/10.3389/fimmu.2019.02732
  • Mangiola, S., Young, N. D., Sternberg, P. W., Strube, C., Korhonen, P. K., Mitreva, M., Scheerlinck, J. P., Hofmann, A., Jex, A. R., & Gasser, R. B. (2014). Analysis of the transcriptome of adult Dictyocaulus filaria and comparison with Dictyocaulus viviparus, with a focus on molecules involved in host-parasite interactions. International Journal for Parasitology, 44(3-4), 251–261. https://doi.org/10.1016/j.ijpara.2013.12.003
  • Manirarora, J. N., Walker, K. E., Patil, V., Renukaradhya, G. J., LaBresh, J., Sullivan, Y., Francis, O., & Lunney, J. K. (2022). Development and characterization of new monoclonal antibodies against porcine interleukin-17A and interferon-gamma. Frontiers in Immunology, 13, 786396. https://doi.org/10.3389/fimmu.2022.786396
  • Papadopoulos E, Sotiraki S, Himonas C, & Fthenakis GC. (2004). Treatment of small lungworm infestation in sheep by using moxidectin. Veterinary Parasitology, 121(3-4), 329–336. https://doi.org/10.1016/j.vetpar.2004.02.026
  • Panayotova-Pencheva, M. S., & Alexandrov, M. T. (2010). Some pathological features of lungs from domestic and wild ruminants with single and mixed protostrongylid infections. Veterinary Medicine International, 2010, 741062. https://doi.org/10.4061/2010/741062
  • Panuska, C. (2006). Lungworms of ruminants. Veterinary Clinics of North America: Food Animal Practice, 22(3), 583–593. https://doi.org/10.1016/j.cvfa.2006.06.002
  • Ram, P. K., Singh, S. K., Kumari, P., Srivastava, M., Sudan, V., Pandey, R. P., & Garg, S. K. (2020). Role of cytokines in the clinical manifestation of exophthalmia in newborn calves with tropical theileriosis. Parasite Immunology, 42(10), e12761.
  • Rehbein, S., Knaus, M., Li, J., Antretter, A., Mayr, S., Visser, M., & Hamel, D. (2022). Treatment of natural Protostrongylus rufescens lungworm infection in sheep with eprinomectin 5 mg/mL topical solution. Veterinary Parasitology, 301, 109639. https://doi.org/10.1016/j.vetpar.2021.109639
  • Rehbein, S., & Visser, M. (2002). Efficacy of ivermectin delivered via a controlled-release capsule against small lungworms (Protostrongylidae) in sheep. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health, 49(7), 313–316. https://doi.org/10.1046/j.1439-0450.2002.00576.x
  • Sağlam, Y. S., Bozoğlu, H., & Baş, A. T. (1998). Erzurum ve çevresinde kuzu enzootik pnömonileri üzerinde bakteriyolojik ve patolojik incelemeler. Etlik Veteriner Mikrobiyoloji Dergisi, 10(1), 1-17.
  • Samar, A. E., Bahaa, E. D., & Shereen, F. M. (2017). Parasitological, pathological and immunological effects of Nigella sativa oil in experimentally Toxocara canis infected mice. Tropical Biomedicine, 34(4), 781–794.
  • Sasai, M., & Yamamoto, M. (2019). Innate, adaptive, and cell-autonomous immunity against Toxoplasma gondii infection. Experimental & Molecular Medicine, 51(12), 1–10. https://doi.org/10.1038/s12276-019-0353-9
  • Ülgen, M., Sönmez, G., & Aydın, F. (1997). Kuzu pnömonileri üzerinde mikrobiyolojik ve histopatolojik incelemeler. Etlik Veteriner Mikrobiyoloji Dergisi, 9(2), 55-70.
  • Zafari, S., Mohtasebi, S., Sazmand, A., Bahari, A., Sargison, N. D., & Verocai, G. G. (2022). The prevalence and control of lungworms of pastoral ruminants in Iran. Pathogens, 11(12), 1392. https://doi.org/10.3390/pathogens11121392
There are 37 citations in total.

Details

Primary Language English
Subjects Veterinary Pathology
Journal Section Research Articles
Authors

Emin Karakurt 0000-0003-2019-3690

Semistan Kızıltepe 0000-0003-3727-8893

Ayfer Yıldız 0000-0002-6569-5435

Early Pub Date November 24, 2023
Publication Date December 15, 2023
Submission Date September 22, 2023
Acceptance Date November 23, 2023
Published in Issue Year 2023 Volume: 8 Issue: 3

Cite

APA Karakurt, E., Kızıltepe, S., & Yıldız, A. (2023). Immunohistochemical evaluation of IFN-γ levels in sheep verminous pneumonia. Journal of Advances in VetBio Science and Techniques, 8(3), 216-225. https://doi.org/10.31797/vetbio.1364749
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