Infant rhesus macaques used in this study, 5–6 months of age, were obtained from Institute of Medical Biology, Chinese Academy of Medical Sciences (IMBCAMS). The study protocol was approved (DWSP201809002) by the Committee on the Ethics of the IMBCAMS and was conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals from the National Research Council of the National Academies and the Guidance for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of the People’s Republic of China (2006). During the study periods, the monkeys were maintained at Animal Biosafety Level 2, housed individually in cages in a climate-controlled room (temperature of 18-25°C and humidity 30-70%) with a 12 h light/dark cycle, received
food and fruits strictly complying with requirements of animal welfare, and had free access to water. After this experiment, the monkeys were confirmed completely recovered from B.p infection, and were put back to Monkey Mountain of IMBCAMS, allowing them to die naturally.
Bacterial Strains and Media
B.p strain 2016-CY-41 used in this study was recently isolated from a patient in China and was obtained from the National Institutes for Food and Drug Control (Beijing, China). The polymorphisms in the pertussis toxin promoter (ptxP), pertussis toxin subunit 1 (ptxA), pertactin (prn), fimbrial (fim)2 and fim3 were assessed by DNA sequencing. The genotype of 2016-CY-41 was ptxP1/ptxA1/prn1/fim2-1/fim3-1. For B.p infection experiments, bacteria were grown on Bordet-Gengou agar (B-G) plates (BG, Hopebio, CHN) containing 20% defibrinated sheep blood (Nanjinglezhen, CHN) for 48 to 72 h at 37°C. Colonies from fresh B-G plates were resuspended in isotonic saline, diluted to a concentration of 1011 CFU/mL using a turbidimetric method, and used within 2 h of preparation. For the culture of nasopharyngeal wash (NPW) bacteria, Regan-Lowe plates, prepared from Regan-Lowe charcoal agar base with 10% defibrinated sheep blood and 40 μg/mL cephalexin (Oxoid, US), were used.
Infection and Transmission in Rhesus Macaques
Seven healthy male rhesus macaques, aged 5 to 6 months and weighing from 1.2-1.8 kg, were obtained from the IMBCAMS (Animal License No. SCXK (Dian) K2015-0004) and randomly assigned into two groups (Table 1). Group 1, containing 5 macaques were challenged with strain 2016-CY-41 via aerosol exposure using an aerosolization apparatus designed by our lab and produced by Lanfang Honlan Equipment Co (Additional file 1). The apparatus was composed of a rectangular Plexiglas chamber with a removable lid (40 cm × 60 cm × 40 cm), a pump and a medical nebulizer (average atomization rate: ³ 0.15 mL/min, working pressure: 60-150 KPa, normal working condition: 10-40°C). The pump was connected to the inlet side of the nebulizer to deliver a B.p suspension for atomization. The outlet side of the nebulizer was connected to two inlet ports of the challenge chamber to deliver atomized B.p to the interior of the chamber. An outlet tube with an air filter was connected to the challenge chamber to remove air. An air sampling port was embedded in the middle of the challenge chamber to monitor the actual concentration of aerosolized B.p inside the chamber. Animals were infected via the challenge chamber for 60 min. Within the 60 min period, the air sample was removed from the sampling port every 10 min for the assessment of the concentration of B.p inside the chamber.
At 2 days post-infection (dpi), 1 challenged macaque was cohoused with 1 naive animal in one cage, and the animals were separated after 4 days to investigate transmission. Thus, 2 macaques who were cohoused with 2016-CY-41-challenged animals formed group 2.
Evaluation of Animals and Sample Collection
A schematic of the specimen collection timeline is displayed in Figure 1. Total white blood cell (WBC) counts were measured by blood cell counting. Coughing frequency was monitored by a recording device, which was reviewed, and the number of coughs at four 30-min periods each day (7:00-7:30 a.m., 10:00-10:30 a.m., 2:00-2:30 p.m., and 8:00-8:30 p.m.) was calculated. The average number of coughs per hour for each day was calculated as the mean for all four observation periods for all animals in each group. The NPW was serially diluted in saline and plated on Regan-Lowe plates. The number of CFUs was calculated after 4-5 days of incubation at 37°C. The B.p colonies were identified by examining the colony morphology and hemolysis on Regan-Lowe plates and by polymerase chain reaction (PCR) amplification of IS481, a genomic insertion site that is specific for B.p.
Detection of Serum Antibodies
Serum was separated, and anti-PT, anti - filamentous hemagglutinin (FHA), anti-PRN and anti- adenylate cyclase toxin (ACT) levels were measured using an enzyme-linked immunosorbent assay (ELISA). A 96-well micro plate was coated with 3 μg/mL of the antigens PT, FHA, PRN, and ACT and incubated at 4°C overnight. Then, the plates were blocked with 3% (w/v) bovine serum albumin (BSA, Amresco, A0332) in phosphate buffered saline (PBS) at 37°C for 2 h. Diluted serum was added to the microplate and incubated at 37°C for 1 h. After washing, horseradish peroxidase (HRP)-labeled sheep anti-monkey immunoglobulin G (IgG) was added to the microplate, and the plate was incubated at 37°C for 1 h. All of the ELISA plates were developed using tetramethylbenzidine (TMB; Solarbio, PR1200) to generate a colorimetric reaction and terminated with 2 mol/L of H2SO4. For each set of ELISA plates, a WHO international standard pertussis antiserum was used as a reference (NIBSC code: 06/140). For anti-ACT, a blank sample was included in each plate, and optical density (OD) values ≥ 2.1-fold those of the blank sample were set as cutoff values (all the antigens were from the Department of DTP Vaccine and Toxin, National Institute for Food and Drug Control, China). Results were presented as geometric mean concentrations (GMCs) or geometric mean titers (GMTs) and their 95% confidence intervals (CIs).
Measurement of Cytokines
Serum concentrations of interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-12/23p40, IL-13, IL-17A, interferon (INF)-γ, and tumor necrosis factor (TNF)-a were detected by the Luminex technique with a Milliplex NHP Magnetic Bead Panel (Merck Millipore, US) according to the manufacturer’s instructions. An unpaired t-test was used to test for differences between the pre-challenge and peak cytokine production post-challenge period (2/4, 6/8, 10/12, 14/16, 21/23, 28/30 dpi, the latter is for the transmission group as Fig. 1 indicated) for each animal due to the highly variable individual starting concentrations between animals and variability of the peak response for each cytokine post-infection.
Data were graphed and analyzed using GraphPad Prism version 7.0 (GraphPad Software, Inc.). The data are presented as the mean ± standard errors of the mean or GMCs / or GMTs and their 95% confidence intervals (CIs). Unpaired student’s t-test was utilized to assess statistical significance.