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Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV)

Yıl 2022, Cilt: 41 Sayı: 1, 43 - 48, 26.07.2022
https://doi.org/10.30782/jrvm.1059876

Öz

Respiratory Syncytial Virus (RSV) is an important cause of sheep and goat respiratory tract disease. RSV usually replicates in the airway epithelium and proinflammatory cytokines and chemokines are induced. In this study, it was aimed to investigate TNF-a expression in natural RSV-infected sheep and goat lung paraffin blocks by immunohistochemical method. The study material consisted of twenty nine lung archive paraffin blocks (nineteen sheep and ten goats), which were admitted to Etlik Veterinary Control and Research Institute from Ankara and surrounding provinces with the suspicion of pneumonia between 2015 and 2020. Histopathological findings such as degeneration and desquamation in the bronchi and bronchial epithelium, fibromuscular hypertrophy, hyperplasia in the peribronchial lymphoid tissue, cell infiltration in the interalveolar septum, and no statistical difference was detected in the sheep and goat lung paraffin block tissues (p> 0.05). Immunohistochemically, RSV replication in bronchial and bronchial epithelium and cell debris, bronchial glands, interalveolar septum inflammatory cells, alveolar macrophages was statistically similar in sheep and goats (p> 0.05). It was determined that the expression of TNF-a was more intensely stained in the lung tissue of goats than sheep (p<0.05).
In conclusion, it was noticed that RSV infections have an important place in respiratory system infections in sheep and goat herds. It is recommended to carry out studies to detect cytokine storm in natural RSV infections in sheep and goats, and to investigate antibody therapy, which is a new treatment method, in sheep and goat viral infections.

Destekleyen Kurum

KÜ-BAP

Proje Numarası

01/2020-2

Teşekkür

This study was supported by the Kastamonu Scientific Research project (Project No: KÜ-BAP 01/2020-2).

Kaynakça

  • 1. Sitthicharoenchai P, Alnajjar S, Ackermann MR. A model of respiratory syncytial virus (RSV) infection of infants in newborn lambs. Cell and tissue research 2020;380:313-324.
  • 2. Alansari H, Potgieter L. Molecular cloning and sequence analysis of the phosphoprotein, nucleocapsid protein, matrix protein and 22K (M2) protein of the ovine respiratory syncytial virus. Journal of General Virology 1994;75:3597-3601.
  • 3. Lehmkuhl H, Smith M, Cutlip R. Morphogenesis and structure of caprine respiratory syncytial virus. Archives of virology 1980;65:269-276. 4. Kast JI, McFarlane AJ, Głobińska A, et al. Respiratory syncytial virus infection influences tight junction integrity. Clinical & Experimental Immunology 2017;190:351-359.
  • 5. Sacco RE, McGill J, Pillatzki AE, et al. Respiratory syncytial virus infection in cattle. Veterinary pathology 2014;51:427-436.
  • 6. Trudel M, Nadon F, Simard C, et al. Comparison of caprine, human and bovine strains of respiratory syncytial virus. Archives of virology 1989;107:141-149.
  • 7. Grubbs ST, Kania SA, Potgieter L. Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay. Journal of veterinary diagnostic investigation 2001;13:128-132.,
  • 8. Baker JC. Human and bovine respiratory syncytial virus: immunopathologic mechanisms. Veterinary Quarterly 1991;13:47-59.
  • 9. Van der Poel W, Brand A, Kramps J, et al. Respiratory syncytial virus infections in human beings and in cattle. Journal of Infection 1994;29:215-228.
  • 10. Meyerholz DK, Grubor B, Fach SJ, et al. Reduced clearance of respiratory syncytial virus infection in a preterm lamb model. Microbes and infection 2004;6:1312-1319.
  • 11. Eleraky NZ, Kania SA, Evermann JF, et al. Comparison of targeting F and G protein genes to detect bovine and ovine respiratory syncytial viruses. Journal of veterinary diagnostic investigation 2003;15:277-280.
  • 12. Eleraky NZ, Kania SA, Potgieter LN. The ovine respiratory syncytial virus F gene sequence and its diagnostic application. Journal of veterinary diagnostic investigation 2001;13:455-461.
  • 13. Bem RA, Domachowske JB, Rosenberg HF. Animal models of human respiratory syncytial virus disease. American Journal of Physiology-Lung Cellular and Molecular Physiology 2011;301:L148-L156.
  • 14. Hägglund S, Svensson C, Emanuelson U, et al. Dynamics of virus infections involved in the bovine respiratory disease complex in Swedish dairy herds. The Veterinary Journal 2006;172:320-328.
  • 15. Johnson JE, Gonzales RA, Olson SJ, et al. The histopathology of fatal untreated human respiratory syncytial virus infection. Modern Pathology 2007;20:108-119.
  • 16. Viuff B, Tjørnehøj K, Larsen LE, et al. Replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus. The American journal of pathology 2002;161:2195-2207.
  • 17. Viuff B, Uttenthal Å, Tegtmeier C, et al. Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization. Veterinary Pathology 1996;33:383-390.
  • 18. Larios Mora A, Detalle L, Van Geelen A, et al. Kinetics of respiratory syncytial virus (RSV) Memphis strain 37 (M37) infection in the respiratory tract of newborn lambs as an RSV infection model for human infants. PLoS One 2015;10:e0143580.
  • 19. Valarcher J-F, Taylor G. Bovine respiratory syncytial virus infection. Veterinary research 2007;38:153-180.
  • 20. Garg R, Latimer L, Wang Y, et al. Maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from RSV in newborn lambs. Vaccine 2016;34:261-269.
  • 21. Panuska JR, Cirino NM, Midulla F, et al. Productive infection of isolated human alveolar macrophages by respiratory syncytial virus. The Journal of clinical investigation 1990;86:113-119.
  • 22. Midulla F, Villani A, Panuska JR, et al. Respiratory syncytial virus lung infection in infants: immunoregulatory role of infected alveolar macrophages. Journal of Infectious Diseases 1993;168:1515-1519.
  • 23. Neuzil KM, Tang Y-W, Graham BS. Protective role of TNF-α in respiratory syncytial virus infection in vitro and in vivo. The American journal of the medical sciences 1996;311:201-204.
  • 24. Cirino NM, Panuska JR, Villani A, et al. Restricted replication of respiratory syncytial virus in human alveolar macrophages. Journal of general virology 1993;74:1527-1537.
  • 25. Merolla R, Rebert NA, Tsiviste PT, et al. Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor alpha and interferon beta. American journal of respiratory and critical care medicine 1995;152:1358-1366.
  • 26. Grubor B, Gallup JM, Meyerholz DK, et al. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs. Clinical and Vaccine Immunology 2004;11:599-607.
  • 27. Yavuz O, Dinçel GÇ. Expression of COX-2, HMGB-1 and CD68 in lung tissue in sheep fibrinous bronchopneumonia. Medycyna Weterynaryjna 2020;76. 28. F Rosenberg H, B Domachowske J. Inflammatory responses to respiratory syncytial virus (RSV) infection and the development of immunomodulatory pharmacotherapeutics. Current medicinal chemistry 2012;19:1424-1431.
  • 29. Guzman E, Taylor G. Immunology of bovine respiratory syncytial virus in calves. Molecular immunology 2015;66:48-56. 30. Becker S, Quay J, Soukup J. Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages. The Journal of Immunology 1991;147:4307-4312.
  • 31. Sow FB, Gallup JM, Olivier A, et al. Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs. American Journal of Physiology-Lung Cellular and Molecular Physiology 2011;300:L12-L24.
  • 32. Rųntved C, Tjųrnehųj K, Viuff B, et al. Increased pulmonary secretion of tumor necrosis factor-a in calves experimentally infected with bovine respiratory syncytial virus. Veterinary Immunology and Immunopathology 2000;76:214.
  • 33. Vilcek S, Elvander M, Ballagi-Pordany A, et al. Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples. Journal of Clinical Microbiology 1994;32:2225-2231.
  • 34. Gershwin LJ. Immunology of bovine respiratory syncytial virus infection of cattle. Comparative immunology, microbiology and infectious diseases 2012;35:253-257.
  • 35. Belknap EB, Ciszewski DK, Baker JC. Experimental respiratory syncytial virus infection in calves and lambs. Journal of Veterinary Diagnostic Investigation 1995;7:285-298.
  • 36. West K, Petrie L, Haines DM, et al. The effect of formalin-inactivated vaccine on respiratory disease associated with bovine respiratory syncytial virus infection in calves. Vaccine 1999;17:809-820.
  • 37. Kimman T, Straver P, Zimmer G. Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: morphologic and serologic findings. American journal of veterinary research 1989;50:684-693.
  • 38. Derscheid RJ, Ackermann MR. Perinatal lamb model of respiratory syncytial virus (RSV) infection. Viruses 2012;4:2359-2378.
  • 39. Krishnadasan B, Naidu BV, Byrne K, et al. The role of proinflammatory cytokines in lung ischemia-reperfusion injury. The Journal of thoracic and cardiovascular surgery 2003;125:261-272.
  • 40. Franke G, Freihorst J, Steinmüller C, et al. Interaction of alveolar macrophages and respiratory syncytial virus. Journal of immunological methods 1994;174:173-184.

Doğal Solunum Sinsitiyal Virüsü (RSV) ile Enfekte Koyun ve Keçi Akciğer Parafin Bloklarında TNF-a Ekspresyonunun İmmünohistokimyasal Olarak İncelenmesi

Yıl 2022, Cilt: 41 Sayı: 1, 43 - 48, 26.07.2022
https://doi.org/10.30782/jrvm.1059876

Öz

Respiratuar Sinsitiyal Virüsü (RSV), koyun ve keçi solunum yolu hastalığının önemli bir nedenidir. RSV, genellikle solunum yolları epitelinde replike olur ve proinflamatuar sitokinler ve kemokinler indüklenir. Yapılan çalışmada, doğal RSV ile enfekte koyun ve keçi akciğer parafin bloklarında TNF-a ekspresyonu immunohistokimyasal yöntem ile araştırılması amaçlandı. Çalışma materyali, Etlik Veteriner Kontrol ve Araştırma Enstitüsüne, Ankara ve çevre illerinden 2015- 2020 yılları arasında pnömoni şüphesi ile gelen yirmi dokuz adet hasta (on dokuz Koyun ve on keçi) akciğer arşiv parafin blokları oluşturdu. Koyun ve keçi akciğer parafin blok dokularında bronş ve bronş epitelinde dejenerasyon ve deskuamasyon, fibromusküler hipertrofi, peribronşiyal lenfoid dokuda hiperplazi, interalveolar septumda hücre infiltrasyonu gibi histopatolojik bulgular istatistiksel olarak anlamlı değildi (p>0.05). İmmunohistaokimyasal olarak, bronş ve bronş epitelinde ve hücre döküntülerinde, bronş bezlerinde, interalveolar septum inflamatuar hücrelerde, alveolar makrofajlarda RSV replikasyonu koyun ve keçilerde istatiksel olarak benzerdi (p> 0.05). TNF-a’nın ekspresyonu keçilerin akciğer dokusunda koyunlara göre daha fazla şiddetli boyandığı (p<0.05) edildiği belirlendi.
Sonuç olarak, koyun ve keçi sürülerinde solunum sistemi enfeksiyonlarında RSV enfeksiyonlarının önemli bir yer tuttuğu fark edildi. Koyun ve keçilerde doğal RSV enfeksiyonlarında sitokin fırtınasının tespit edilmesine yönelik çalışmaların yapılmasına, ayrıca günümüzde yeni bir tedavi yöntemi olan antikor tedavisinin koyun ve keçi viral enfeksiyonlarında araştırılması önerilmektedir.

Proje Numarası

01/2020-2

Kaynakça

  • 1. Sitthicharoenchai P, Alnajjar S, Ackermann MR. A model of respiratory syncytial virus (RSV) infection of infants in newborn lambs. Cell and tissue research 2020;380:313-324.
  • 2. Alansari H, Potgieter L. Molecular cloning and sequence analysis of the phosphoprotein, nucleocapsid protein, matrix protein and 22K (M2) protein of the ovine respiratory syncytial virus. Journal of General Virology 1994;75:3597-3601.
  • 3. Lehmkuhl H, Smith M, Cutlip R. Morphogenesis and structure of caprine respiratory syncytial virus. Archives of virology 1980;65:269-276. 4. Kast JI, McFarlane AJ, Głobińska A, et al. Respiratory syncytial virus infection influences tight junction integrity. Clinical & Experimental Immunology 2017;190:351-359.
  • 5. Sacco RE, McGill J, Pillatzki AE, et al. Respiratory syncytial virus infection in cattle. Veterinary pathology 2014;51:427-436.
  • 6. Trudel M, Nadon F, Simard C, et al. Comparison of caprine, human and bovine strains of respiratory syncytial virus. Archives of virology 1989;107:141-149.
  • 7. Grubbs ST, Kania SA, Potgieter L. Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay. Journal of veterinary diagnostic investigation 2001;13:128-132.,
  • 8. Baker JC. Human and bovine respiratory syncytial virus: immunopathologic mechanisms. Veterinary Quarterly 1991;13:47-59.
  • 9. Van der Poel W, Brand A, Kramps J, et al. Respiratory syncytial virus infections in human beings and in cattle. Journal of Infection 1994;29:215-228.
  • 10. Meyerholz DK, Grubor B, Fach SJ, et al. Reduced clearance of respiratory syncytial virus infection in a preterm lamb model. Microbes and infection 2004;6:1312-1319.
  • 11. Eleraky NZ, Kania SA, Evermann JF, et al. Comparison of targeting F and G protein genes to detect bovine and ovine respiratory syncytial viruses. Journal of veterinary diagnostic investigation 2003;15:277-280.
  • 12. Eleraky NZ, Kania SA, Potgieter LN. The ovine respiratory syncytial virus F gene sequence and its diagnostic application. Journal of veterinary diagnostic investigation 2001;13:455-461.
  • 13. Bem RA, Domachowske JB, Rosenberg HF. Animal models of human respiratory syncytial virus disease. American Journal of Physiology-Lung Cellular and Molecular Physiology 2011;301:L148-L156.
  • 14. Hägglund S, Svensson C, Emanuelson U, et al. Dynamics of virus infections involved in the bovine respiratory disease complex in Swedish dairy herds. The Veterinary Journal 2006;172:320-328.
  • 15. Johnson JE, Gonzales RA, Olson SJ, et al. The histopathology of fatal untreated human respiratory syncytial virus infection. Modern Pathology 2007;20:108-119.
  • 16. Viuff B, Tjørnehøj K, Larsen LE, et al. Replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus. The American journal of pathology 2002;161:2195-2207.
  • 17. Viuff B, Uttenthal Å, Tegtmeier C, et al. Sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization. Veterinary Pathology 1996;33:383-390.
  • 18. Larios Mora A, Detalle L, Van Geelen A, et al. Kinetics of respiratory syncytial virus (RSV) Memphis strain 37 (M37) infection in the respiratory tract of newborn lambs as an RSV infection model for human infants. PLoS One 2015;10:e0143580.
  • 19. Valarcher J-F, Taylor G. Bovine respiratory syncytial virus infection. Veterinary research 2007;38:153-180.
  • 20. Garg R, Latimer L, Wang Y, et al. Maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from RSV in newborn lambs. Vaccine 2016;34:261-269.
  • 21. Panuska JR, Cirino NM, Midulla F, et al. Productive infection of isolated human alveolar macrophages by respiratory syncytial virus. The Journal of clinical investigation 1990;86:113-119.
  • 22. Midulla F, Villani A, Panuska JR, et al. Respiratory syncytial virus lung infection in infants: immunoregulatory role of infected alveolar macrophages. Journal of Infectious Diseases 1993;168:1515-1519.
  • 23. Neuzil KM, Tang Y-W, Graham BS. Protective role of TNF-α in respiratory syncytial virus infection in vitro and in vivo. The American journal of the medical sciences 1996;311:201-204.
  • 24. Cirino NM, Panuska JR, Villani A, et al. Restricted replication of respiratory syncytial virus in human alveolar macrophages. Journal of general virology 1993;74:1527-1537.
  • 25. Merolla R, Rebert NA, Tsiviste PT, et al. Respiratory syncytial virus replication in human lung epithelial cells: inhibition by tumor necrosis factor alpha and interferon beta. American journal of respiratory and critical care medicine 1995;152:1358-1366.
  • 26. Grubor B, Gallup JM, Meyerholz DK, et al. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs. Clinical and Vaccine Immunology 2004;11:599-607.
  • 27. Yavuz O, Dinçel GÇ. Expression of COX-2, HMGB-1 and CD68 in lung tissue in sheep fibrinous bronchopneumonia. Medycyna Weterynaryjna 2020;76. 28. F Rosenberg H, B Domachowske J. Inflammatory responses to respiratory syncytial virus (RSV) infection and the development of immunomodulatory pharmacotherapeutics. Current medicinal chemistry 2012;19:1424-1431.
  • 29. Guzman E, Taylor G. Immunology of bovine respiratory syncytial virus in calves. Molecular immunology 2015;66:48-56. 30. Becker S, Quay J, Soukup J. Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages. The Journal of Immunology 1991;147:4307-4312.
  • 31. Sow FB, Gallup JM, Olivier A, et al. Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs. American Journal of Physiology-Lung Cellular and Molecular Physiology 2011;300:L12-L24.
  • 32. Rųntved C, Tjųrnehųj K, Viuff B, et al. Increased pulmonary secretion of tumor necrosis factor-a in calves experimentally infected with bovine respiratory syncytial virus. Veterinary Immunology and Immunopathology 2000;76:214.
  • 33. Vilcek S, Elvander M, Ballagi-Pordany A, et al. Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples. Journal of Clinical Microbiology 1994;32:2225-2231.
  • 34. Gershwin LJ. Immunology of bovine respiratory syncytial virus infection of cattle. Comparative immunology, microbiology and infectious diseases 2012;35:253-257.
  • 35. Belknap EB, Ciszewski DK, Baker JC. Experimental respiratory syncytial virus infection in calves and lambs. Journal of Veterinary Diagnostic Investigation 1995;7:285-298.
  • 36. West K, Petrie L, Haines DM, et al. The effect of formalin-inactivated vaccine on respiratory disease associated with bovine respiratory syncytial virus infection in calves. Vaccine 1999;17:809-820.
  • 37. Kimman T, Straver P, Zimmer G. Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: morphologic and serologic findings. American journal of veterinary research 1989;50:684-693.
  • 38. Derscheid RJ, Ackermann MR. Perinatal lamb model of respiratory syncytial virus (RSV) infection. Viruses 2012;4:2359-2378.
  • 39. Krishnadasan B, Naidu BV, Byrne K, et al. The role of proinflammatory cytokines in lung ischemia-reperfusion injury. The Journal of thoracic and cardiovascular surgery 2003;125:261-272.
  • 40. Franke G, Freihorst J, Steinmüller C, et al. Interaction of alveolar macrophages and respiratory syncytial virus. Journal of immunological methods 1994;174:173-184.
Toplam 37 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Veteriner Cerrahi
Bölüm Research Articles
Yazarlar

Funda Terzi 0000-0002-6184-5408

Yavuz Ulusoy

Bahadır Kılınç 0000-0003-3426-2116

Ayşe Gül Dal 0000-0002-0607-2182

Rabi Salik 0000-0002-0364-697X

Proje Numarası 01/2020-2
Yayımlanma Tarihi 26 Temmuz 2022
Kabul Tarihi 16 Mayıs 2022
Yayımlandığı Sayı Yıl 2022 Cilt: 41 Sayı: 1

Kaynak Göster

APA Terzi, F., Ulusoy, Y., Kılınç, B., Dal, A. G., vd. (2022). Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV). Journal of Research in Veterinary Medicine, 41(1), 43-48. https://doi.org/10.30782/jrvm.1059876
AMA Terzi F, Ulusoy Y, Kılınç B, Dal AG, Salik R. Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV). J Res Vet Med. Temmuz 2022;41(1):43-48. doi:10.30782/jrvm.1059876
Chicago Terzi, Funda, Yavuz Ulusoy, Bahadır Kılınç, Ayşe Gül Dal, ve Rabi Salik. “Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected With Natural Respiratory Syncytial Virus (RSV)”. Journal of Research in Veterinary Medicine 41, sy. 1 (Temmuz 2022): 43-48. https://doi.org/10.30782/jrvm.1059876.
EndNote Terzi F, Ulusoy Y, Kılınç B, Dal AG, Salik R (01 Temmuz 2022) Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV). Journal of Research in Veterinary Medicine 41 1 43–48.
IEEE F. Terzi, Y. Ulusoy, B. Kılınç, A. G. Dal, ve R. Salik, “Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV)”, J Res Vet Med, c. 41, sy. 1, ss. 43–48, 2022, doi: 10.30782/jrvm.1059876.
ISNAD Terzi, Funda vd. “Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected With Natural Respiratory Syncytial Virus (RSV)”. Journal of Research in Veterinary Medicine 41/1 (Temmuz 2022), 43-48. https://doi.org/10.30782/jrvm.1059876.
JAMA Terzi F, Ulusoy Y, Kılınç B, Dal AG, Salik R. Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV). J Res Vet Med. 2022;41:43–48.
MLA Terzi, Funda vd. “Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected With Natural Respiratory Syncytial Virus (RSV)”. Journal of Research in Veterinary Medicine, c. 41, sy. 1, 2022, ss. 43-48, doi:10.30782/jrvm.1059876.
Vancouver Terzi F, Ulusoy Y, Kılınç B, Dal AG, Salik R. Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV). J Res Vet Med. 2022;41(1):43-8.