Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, especially the co-pathologies between RSV and Spn, is known.  Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups were mock nebulized. At day three post-RSV infection, one RSV group (RSV/Spn) and one mock-nebulized group (Spn only) were inoculated with Spn intratracheally. At day six post-RSV infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/Spn trended with higher RSV titers, but lower Spn. Additionally, lung lesions were observed to be more intense in the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts.

Experimental Design:

Animals: A total of 20, 2-3 day-old, colostrum-deprived lambs, were randomly divided into four groups with five animals per group: RSV only, RSV-Spn co-infection, Spn only, and uninfected control. Animals were group-housed to reduce stress and colostrum as withheld to reduce any interference with cross-reactive anti-ovine RSV immune responses passing to the lamb. We do not envision that this would alter their immune responses to the virus. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the USDA. Animal use was approved by the Institutional Animal Care and Use Committee of Iowa State University.  All experiments were performed following relevant guidelines and regulations as set by regulatory bodies. 

Infectious Agents: Lambs were infected with RSV strain M37, purchased from Meridian BioSciences (Memphis, TN, USA). This strain is a wild type A RSV isolated from the respiratory secretions of an infant hospitalized for bronchiolitis [35, 36]. M37 was grown in HELA cells and stored at -80°C in media containing 20% sucrose [37]. 6 mL of 1.27 x 10⁷IFFU/mL in media containing 20% sucrose or cell-conditioned mock media (also containing 20% sucrose) was nebulized using PARI LC Sprint™ nebulizers to each lamb over 25-30 minutes resulting in the total inhalation of about 3 mL by each lamb [37]. Spn serotype 22F was grown overnight at 37°Cin Todd Hewitt media containing 2% yeast extract, 50 mg/ml of gentamicin, and 10% bovine serum. Colony-forming units (CFUs) were calculated by OD₆₀₀ with confirmation by dilution plating on Tryptic Soy Agar (TSA) plates with 5% sheep blood containing gentamicin.

Infections: For viral inoculations, infectious focus forming units (IFFU), where only replication-competent virus is detected by antibody in limiting dilution assays, were utilized. Two groups were exposed to nebulized RSV M37 (1.27x107 IFFU/mL), as done previously [37, 38], on day 0. One of the RSV infected groups was inoculated intratracheally with 2 ml normal saline as a mock Spn infection (RSV group) using syringe and needle, while the second RSV-infected group was inoculated intratracheally with 2 ml solution containing Spn serotype 22F (2x106 CFU/ml) 3 days post-RSV nebulization (RSV-Spn group). The other two groups were exposed to nebulized cell-conditioned mock media containing 20% sucrose at day 0 and inoculated intratracheally with either normal saline (control group) or solution containing Spn (2x106 CFU/ml) at day three post nebulization (Spn group). At day six post-RSV infection, all lambs were humanely euthanized with Fatal Plus immediately before necropsy. 

Gross Pathology: An autopsy was performed to evaluate the macroscopic lung lesions. After removal, each lung was examined by a pathologist similar to prior studies [38, 39]. If lesions were present, percentage involvement was estimated for each lung lobe. Percentages were converted to a scale using the following formula: 0%=0, 1-9%=1, 10-39%=2, 40-69%=3, 70-100%=4.  Group averages were calculated for the gross lesion score. Lung samples were collected, including sterile lung tissue for bacterial isolation, frozen lung sample for RT-qPCR, bronchioalveolar lavage fluid (BALF) from right caudal lung lobe for RSV IFFU assay and RT-qPCR, and lung pieces from different lobes were fixed in 10% neutral buffered formalin for histological assessment. 

Clinical Observations: Animals were observed three times daily and scored (1-5 on severity) by blinded animal caretakers concerning clinical symptoms including wheezing, lethargy, coughing, nasal/eye discharge while also taking a daily rectal temperature. The animal caretakers were all experienced with RSV infections in lambs from prior studies; however, this was the first time this scoring system has been used. The specific end-point for the study was six days post-RSV infection or if an animal appeared in respiratory distress, failed to take milk replacement at feeding times for longer than 24 hours, or were lethargic. Two animals were euthanized during the study before the six days after meeting the above-stated end-points due to sepsis. 

Lung RSV Viral and Spnbacterial Titers: BALF collected from the right caudal lobe at necropsy by flushing the caudal lobe with 5 mL of cold DMIM and collected back several times as done previously [37, 38]. Collected BALF was used to evaluate RSV IFFU (Plaque assay that counts the number of syncytial cells formed due to viral infection detected by fluid fluorescent antibody technique). BALF was spun for 5 minutes at 3,000g to pellet large debris. Supernatants were  spun through 0.45 am Costar SPIN-X filters (microcentrifuge 15,600g) for 5 minutes. The resulting BALF samples were applied to HELA cells grown to 70% confluence in 12-well culture plates (Fisher Scientific, Hanover Park, IL) at full strength, and three serial dilutions (1:10, 1:100, and 1:1000); all samples were tested in triplicate to determine the viral titer. Plates were stained with a fluorescent antibody technique and as described previously [37, 38]. 100 μL of the right caudal lobe BALF was added to 1 mL TRIzol (Invitrogen) and kept at – 80 °C for the qRT-PCR assay to assess RSV mRNA. Sterile lung tissue samples were used to determine Spntiter. Lung tissue samples were placed in 500μl of sterile PBS and were mechanically homogenized by a pestle. Lung homogenates were pelleted at 100xg, for 5 minutes. Supernatants were serially diluted and applied to 5% sheep blood TSA plates containing gentamycin.

Immunohistochemistry (IHC): Formalin-fixed paraffin-embedded tissue sections were used for IHC, which was performed according to a previously published protocol in our laboratory [32, 37]. Briefly, after deparaffinization and rehydration, antigen retrieval was performed in 10mM TRIZMA base (pH 9.0), 1mM EDTA buffer, and 0.05% Tween 20 with boiling under pressure for up to 15 minutes. Polyclonal goat anti-RSV antibody (Millipore/Chemicon, Temecula, CA; Cat. No. AB1128) was used as the primary antibody after two blocking steps. The first blocking was with 3% bovine serum albumin in Tris-buffered saline +0.05% Tween 20 (TBS-T), and the second was 20% normal swine serum in TBS-T for 15 minutes each. The primary antibody was followed by applying a biotinylatedrabbit anti-goat secondary antibody (KP&L; Cat. No. 16-13-06). Signal development was accomplished using a 1:200 dilution of streptavidin-horseradish peroxidase (Invitrogen; Cat. No. 43-4323) for 30 minutes, followed by incubation with Nova Red chromagen solution (Vector; Cat. No. SK-4800). A positive signal was quantified in both bronchioles and alveoli for each tissue section, and a score of 0-4 was assigned according to an integer-based scale of:  0=no positive alveoli/bronchioles, 1=1-10 positive alveoli/bronchioles, 2=11-39 positive alveoli/bronchioles, 3=40-99 positive alveoli/bronchioles, 4=>100 positive alveoli/bronchioles. IHC for Spnwas performed using a rabbit anti-Streptococcus pneumoniaepolyclonal antibody (Thermofisher Scientific cat. # PA-7259) followed by biotin-labeled goat anti-rabbit IgG antibody (Thermo Fisher Scientific Cat.#: 65-6140). Five random images were taken for each tissue section that was then analyzedby the quantitative Halo program.

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR): BALF and lung tissue homogenates in Trizol were used to assess RSV mRNA expression by RT-qPCR. The assay was performed as published previously in our laboratory [32, 38, 39]. Briefly, RNA isolation from lung tissue and BALF was performed using the TRIzol method followed by standard DNase treatment. RT-qPCR was carried out using the One-Step Fast qRT-PCR Kit master mix (Quanta, BioScience, Gaithersburg, MD) in a StepOnePlus™ qPCR machine (Applied Biosystems, Carlsbad, CA) in conjunction with PREXCEL-Q assay-optimizing calculations. Primers and probe for RSV M37 nucleoprotein were designed based on RSV accession number M74568. Forward primer: 5′-GCTCTTAGCAAAGTCAAGTTGAACGA; reverse primer: 5′-TGCTCCGTTGGATGGTGTATT; hydrolysis probe: 5′-6FAM-ACACTCAACAAAGATCAACTTCTGTCATCCAGC-TAMRA.

Additionally, PBMCs were harvested at six days post-RSV infection and added to RNA later (Sigma) and stored at -80 degrees after an overnight incubation at 4 degrees C. RNA was then isolated by an RNA plus isolation kit (Qiagen, Gaithersburg, MD) per the manufacturer's directions and then subjected to qRT-PCR using a single-step reaction using Luna reagent (NEB, Ipswhich MA). The primers and probes (5'-6FAM and Iowa Black Quencher) used were for IL-10, IFNg, Actin, IL-1b, and IL-17a designed using published lamb cytokine sequences and PrimerDesign (UK) to find optimal pairs.  For the detection of changes in gene expression (normalized on Actin), the RNA levels for each were compared with the levels in uninfected lambs (calibrators), and data are presented as the change in expression of each gene. The ΔC_Tvalue for the tissue sample from the calibrator was then subtracted from the ΔC_Tvalue of the corresponding lung tissue of infected mice (ΔΔC_T). The increase in cytokine mRNA levels in the infected animals' lung tissue samples compared to tissue samples of baseline (calibrator) animals was then calculated as follows: increase = 2^ΔΔCT.

Hematoxylin-Eosin Staining and Histological Scoring of Lung Sections: Hematoxylin-eosin stained sections were examined via a light microscope. The author MA is a licensed veterinary pathologist and scored each section blinded. An integer-based score of 0-4 was assigned for each parameter (bronchiolitis, syncytial cells, epithelial necrosis, epithelial hyperplasia, alveolar septal thickening, neutrophils in the bronchial lumen, neutrophils in the alveolar lumen, alveolar macrophages, peribronchial lymphocytic infiltration, perivascular lymphocytic infiltration, lymphocytes in alveolar septa, fibrosis), with four as the highest score. A final score was calculated by adding up all measured scores to form a 0-48 score, with 48 as the highest, which is called the accumulative histopathological lesion score. We have used this scoring system in several prior publications [38, 40], although use in Spn infected or dually infected lambs was a first for this scoring system. Some of the lambs have larger/bigger normal structures such as the alveolar wall thickness that may be due to the age or the nebulization. Thus, we included these in our score and considered them the minimum score rather than subtracting the score to show zero for the control.

Dual Co-Localization Studies: Hela and Vero cells were infected with RSVA2 (MOI of 0.05) expressing mKate2 fluorescent reporter for 24 hours. Media was washed and replaced with DMEM without antibiotics and labeled Spn (serotypes 6c, 19A, and 22F) similar to (Verhoeven et al., 2014) was added for an additional 4 hours at 37 degrees before washing with PBS and fixing using 2% paraformaldehyde. A Zoe fluorescent microscope was used to randomly document both pathogens on the cells in at least ten fields, with all setting similar overlapping the red and green channels on the brightfield.

RSV Infection of Sheep Neutrophils: Sheep neutrophils were obtained by Ficoll gradient centrifugation with removal of PBMCs. Neutrophil/blood pellets were then lysed in ACK lysis for 5 minutes on ice, followed by washing in PBS. Neutrophils were then resuspended in DMEM 10% and infected with RSVa 2001 at MOI of 1 for 4 hours. Neutrophils were then washed three times and held in RNAlater until qRT-PCR for RSV F transcripts could be performed. For confirmation, we derived neutrophils from adult lambs after Percol centrifugation. We then fixed and permeabilized the neutrophils 4 hours post-infection (MOI of 1 of RSVa 2001 strain) staining with anti-RSV polyclonal antibody (Thermofisher) followed by anti-goat Alexa 555 (Thermofisher). Images were obtained with a ZOE (Biorad, Hercules CA) fluorescent microscope.

Statistical Analysis: Statistical analysis used the Wilcoxon signed-rank test for nonparametric and parameters such as accumulative microscopiclesion scoring, followed by nonparametric comparisons for each pair using the Wilcoxon method. One-way ANOVA was followed by all pairs comparisonby the Tukey-Kramer HSD method for gross lesion scores and viral titer analysesby RT-qPCR and IFFU assays.