Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-γ as a Key Player of the Enhanced Innate Immune Response in Lungs

Abstract

Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-γ) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-γ. IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-γ gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases.

Keywords: Mycoplasma bovis; bovine respiratory disease; cattle; coinfection; immune response; influenza D virus.

FIG 1

Experimental design. (A) Twenty-nine calves were isolated in four separate rooms. Eight calves were inoculated by nebulization with 10⁷ TCID₅₀ of influenza virus D/bovine/France/5920/2014 or 10¹⁰ CFU of Mycoplasma bovis RM16 or combined pathogens. In the control group, five animals received DMEM medium. (B) Timeline and types of samples collected. *, day when sampling or clinical observation was performed.

FIG 2

The coinfection increases the clinical scores. (A) Mean clinical scores with significant differences between groups. (B) Mean accumulated clinical score (ACS) for each group.

FIG 3

Types of macroscopic lesions observed. Macroscopic lesions in respiratory organs in infected compared to control animals. (A) Lung without macroscopic lesions of control calf 9713. (B) Thirty percent of the cranial and accessory lobes of the coinfected animal 9239 had atelectasis (arrows). (C) No gross lesions were observed at 6 dpi on the trachea of calf 9243 infected by M. bovis. (D) Tracheitis lesions with a fibrinopurulent exudate were observed on the mucosal surface of coinfected calf 9718 at 6 dpi.

FIG 4

The coinfection induced greater lesions in the respiratory organs than did monoinfection. Hematoxylin and eosin staining of nasal turbinate (top), trachea (middle), and lung (bottom) tissue samples at 6 dpi. Magnification, ×200; scale bars, 100 μm. H&E-stained sections demonstrating a loss of ciliature and necrosis and exfoliation of the superficial mucosal epithelium (stars), an infiltration of the lamina propria by mononuclear cells (arrows), subacute bronchointerstitial pneumonia with neutrophils in bronchial lumens (asterisk), and neutrophilic and macrophagic alveolitis and peribronchial and septal lymphoplasmocytic infiltration (arrowheads).

FIG 5

The coinfection increases the microscopic lesions. Mean and individual histologic lesion scores in nasal cavity, trachea, and lung for each infected group at 6 dpi.

FIG 6

IDV infection promotes M. bovis colonization of the upper and lower respiratory tracts. Virus titers (top) and bacterial titers (bottom) in nasal swab (left) and bronchoalveolar lavage fluid (right) samples from calves at 0 to 20 dpi and 0 to 14 dpi, respectively. Data represent the mean values ± SEM of IDV RNA and M. bovis DNA copies (log₁₀/ml) measured by RT-qPCR and qPCR, respectively. The number of positive animals/total number of animals is indicated in each bar. The titrations of the IDV (10.7 log₁₀ RNA copies/ml) and M. bovis (11 log₁₀ DNA copies/ml) inoculums were obtained on day zero just before they were administered to the calves by nebulization. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 7

IDV induced a rapid host humoral response in mono- and coinfected calves. Total antibody, IgG1, and IgA titers from serum (A), nasal swab (B), and bronchoalveolar lavage fluid (C) samples from five calves per group were measured by HI assay with influenza virus D/bovine/France/5920/2014 for the IDV-specific antibodies and IDV-specific indirect ELISAs for IgG1 and IgA. Values are presented as means ± SEM of the percent positivity (PP). *, P ≤ 0.05 and ***, P ≤ 0.001 for statistically significant differences between IDV-infected and coinfected groups using Bonferroni’s multiple-comparison test.

FIG 8

The coinfection increased the innate immune response in BAL fluid samples. Fold changes in the mRNA expression (Fluidigm) of 52 bovine genes from BAL fluid samples of mono- or coinfected calves. The fold changes in mRNA expression were calculated for pairs of groups at 2, 7, and 14 dpi. Positive fold changes are colored in red, and negative fold changes in green. The fold change values of differentially expressed genes (DEGs) with a significant difference (P < 0.05) are presented.

FIG 9

The coinfection increased the IFN-γ synthesis in BAL fluid samples. Bovine IFN-γ levels were measured in BAL fluid samples by sandwich ELISA. Data are presented as mean values ± SEM for the cytokine concentrations (pg/ml). *, P ≤ 0.05 for statistical difference between control and coinfected groups using Bonferroni’s multiple-comparison test.