Bovine respiratory syncytial virus (BRSV) is a single negative-stranded RNA virus belonging to the Paramyxoviridae family and shows a close genetic relationship with human respiratory syncytial virus (HRSV). BRSV is the primary etiological agent of respiratory disease in calves aged up to 12 months [1]; beef and dairy cattle worldwide [2, 3]. Initial infection by BRSV alter bovine immune system and facilitates secondary infection of the lower respiratory tract by bacteria [1, 4]. Therefore, BRSV is considered to be a causative agent of bovine respiratory disease complex, which results in economic losses to farmers because of the morbidity and mortality in cattle [2, 3]. According to our preliminary findings based on the gene detection from respiratory samples, paired virus and bacteria were detected; Pasteurella multocida (PM) was the most common bacterial agent (unpublished data). PM is common in the nasopharynx of cattle [5, 6], although PM appears to be part of the normal flora, it can contribute to pneumonia when cattle stressed and/or infected by a respiratory virus [7]. However, the interactions between multiple agents associated with BRDC are not clear. Therefore, the aim of this study was to investigate the effect of BRSV infection on PM adherence to respiratory epithelial cells

Goto, Y

Kobayashi, I

Kubo, M

Mekata, H

Nakamura, K

Okabayashi, T

Sekiguchi, S

Subangkit, M

Sudaryatma, P E

English

Hemera Zoa

 338 | Proc. of the 20th FAVA CONGRESS & The 15th KIVNAS PDHI, Bali Nov 1-3,  2018 Oral Presentation (JSPS-7)  Bovine Respiratory Syncytial Virus Infection Enhances Pasteurella multocida Adherence on Respiratory Epithelial Cells  P. E. Sudaryatma1,2, K. Nakamura2, H. Mekata3,4, S. Sekiguchi2,3, M. Kubo5, I. Kobayashi6, M. Subangkit1,2,   Y. Goto2,3, T. Okabayashi2,3,*)  1Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki, Japan 2Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan 3Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan 4Organization for Promotion of Tenure Track University of Miyazaki, Miyazaki, Japan 5Miyakonojo Meat Inspection Center Miyazaki Prefecture Government, Miyazaki, Japan 6Sumiyoshi education farm, University of Miyazaki, Miyazaki, Japan *)Corresponding author’s email: okbys81@cc.miyazaki-u.ac.jp  Keywords: adherent bacteria, BRDC, BRSV, co-infection.   INTRODUCTION Bovine respiratory syncytial virus (BRSV) is a single negative-stranded RNA virus belonging to the Paramyxoviridae family and shows a close genetic relationship with human respiratory syncytial virus (HRSV). BRSV is the primary etiological agent of respiratory disease in calves aged up to 12 months [1]; beef and dairy cattle worldwide [2, 3]. Initial infection by BRSV alter bovine immune system and facilitates secondary infection of the lower respiratory tract by bacteria [1, 4]. Therefore, BRSV is considered to be a causative agent of bovine respiratory disease complex, which results in economic losses to farmers because of the morbidity and mortality in cattle [2, 3]. According to our preliminary findings based on the gene detection from respiratory samples, paired virus and bacteria were detected; Pasteurella multocida (PM) was the most common bacterial agent (unpublished data). PM is common in the nasopharynx of cattle [5, 6], although PM appears to be part of the normal flora, it can contribute to pneumonia when cattle stressed and/or infected by a respiratory virus [7]. However, the interactions between multiple agents associated with BRDC are not clear. Therefore, the aim of this study was to investigate the effect of BRSV infection on PM adherence to respiratory epithelial cells.  MATERIALS AND METHODS Cell culture, virus and bacteria. A549, HEp-2, MDBK cells were used. Cells were seeded at 1 × 105 cells/ml and cultured as a monolayer in DMEM, 10% FBS; 100 U/ml penicillin and 100 μg/ml streptomycin, maintained at 37°C/5% CO2. BRSV RS-52 strain, propagated in HEp-2 cells and virus titer was checked in a plaque assay using MDBK cells. BRSV was inactivated by UV treatment for 1 h and confirmed in a plaque assay. PM 2368 strain, capsular type B isolated, was grown at 37°C in Brucella broth. BRSV and PM were aliquoted, and kept at –80°C until use.  Adherence assay. A549, HEp-2, and MDBK cells were seeded, >80% confluence, inoculated with BRSV (MOI = 0.1 or 1), UV-inactivated BRSV (MOI = 1), and culture medium as a negative control. Inoculation time was 2 h and replaced with culture medium with 2% FBS. After 24, 48 and 72 h post infection (hpi), BRSV-infected and uninfected cells were exposed with PM suspension for 2 h. Cells were dissociated by trypsinization and collected by centrifugation. Dissociated resulting bacteria plated on Brucella agar. Adherence of bacteria per cell was calculated from total counting of CFU with the total number of monolayer cell. qRT-PCR of mRNA encoding cytokines and chemokines. Cellular RNA was extracted from the cell lysates and performed using a One Step SYBR PrimeScript plus RT-PCR kit; primer sets and amplification conditions for of IL-1, IL-6, IL-8, MCP-1, and RANTES mRNA were described previously [8]. Amplification was carried out in a LightCycler 96 system. Data normalized with GAPDH and calculated using the 2-ΔΔCT method and expressed as a -fold change.  RESULT AND DISCUSSION BRSV infection led to a significant increase in the number of PM cells adhering to respiratory and/or bovine epithelial cells: adherence was both time- and MOI-dependent (Fig. 1 and Fig. 2). Increased adhesion may be related to virus-induced changes in expression of receptors for bacteria depend on types of cell. Here, we found that epithelial cells exposed to PM showed increased expression of genes encoding IL-1, IL-6, IL-8, MCP-1, and RANTES (Fig. 3); however, expression increased further upon co-infection with BRSV. We assume that upregulation of these  Proc. of the 20th FAVA CONGRESS & The 15th KIVNAS PDHI, Bali Nov 1-3,  2018 | 339 cytokines depends on the number of PM cells adhering to the respiratory epithelial cells. These cytokines play important roles in BRDC-related pneumonia.   Fig. 1: The number of PM cells adhering to BRSV-infected A549, HEp-2 and MDBK at 48 and 72 hpi (straight line) was significantly higher than the number adhering to uninfected cells (dash line) at the same times.  * p < 0.01.   Fig. 2: Adherence of Pasteurella multocida (PM) to bovine respiratory syncytial virus (BRSV)-infected epithelial cells is MOI-dependent (closed bars). This was not the case for uninfected cells (open bars) and cells infected with UV-inactivated BRSV (UV-BRSV; patterned bars). * p < 0.05 and ** p < 0.01.   Fig. 3:Expression of IL-1b, IL-6, and IL-8 by PM-infected or BRSV plus PM-infected A549 cells is shown. * p < 0.01.   CONCLUSION We showed here that BRSV infection of respiratory epithelial cells increases adherence of PM, leading to increased expression of genes encoding proinflammatory cytokines. The in vitro data suggest that initial infection by BRSV increases PM adherence, which in turn induces a local acute inflammatory response during the early stages of BRDC  ACKNOWLEDGMENTS This work was supported by the JSPS KAKENHI (Grant Number 17K08080) and JSPS Core-to-core Program, B. Asia-Africa Science Platform. The authors are also grateful to Dr. Uemura (Laboratory of Animal Health, Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki) for providing PM. REFERENCES [1] Gershwin LJ, Berghaus LJ, Arnold K, et al (2005) Immune mechanisms of pathogenetic synergy in concurrent bovine pulmonary infection with Haemophilus somnus and bovine respiratory syncytial virus. Vet Immunol Immunopathol 107:119–130 . doi: 10.1016/j.vetimm.2005.04.004 [2] Beaudeau F, Ohlson  a, Emanuelson U (2010) Associations between bovine coronavirus and bovine respiratory syncytial virus infections and animal performance in Swedish dairy herds. J Dairy Sci 93:1523–1533 . doi: 10.3168/jds.2009-2511 [3] Klima CL, Zaheer R, Cook SR, et al (2014) Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements. J Clin Microbiol 52:438–448 . doi: 10.1128/JCM.02485-13 [4] Larsen LE, Tegtmeier C, Pedersen E (2001) Bovine respiratory syncytial virus (BRSV) pneumonia in beef calf herds despite vaccination. Acta Vet Scand 42:113–121. . doi: 10.1186/1751-0147-42-113 [5] Holman DB, Timsit E, Amat S, et al (2017) The nasopharyngeal microbiota of beef cattle before and after transport to a feedlot. BMC Microbiol 17: . doi: 10.1186/s12866-017-0978-6 [6] Timsit E, Workentine M, Schryvers AB, et al (2016) Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot. Vet Microbiol 187:75–81 . doi: 10.1016/j.vetmic.2016.03.020 [7] Dabo SM, Taylor JD, Confer  a W (2007) Pasteurella multocida and bovine respiratory disease. Anim Health Res Rev 8:129–150 . doi: 10.1017/S1466252307001399 [8] Lau SKP, Lau CCY, Chan K-H, et al (2013) Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment. J Gen Virol 94:2679–2690 . doi: 10.1099/vir.0.055533-0    Figure 1.   Figure 2.   Figure 3.   Figure 3.   

JSPS-7 Bovine Respiratory Syncytial Virus Infection Enhances Pasteurella multocida Adherence on Respiratory Epithelial Cells

Scientific Journals of Bogor Agricultural University

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JSPS-7 Bovine Respiratory Syncytial Virus Infection Enhances Pasteurella multocida Adherence on Respiratory Epithelial Cells

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