From 2006 to 2009, 3,546 nasal and tracheal swab samples were collected for influenza surveillance in four main swine industrial provinces in China (Beijing, Fujian, Guangdong and Shandong). Twenty-nine strains of swine influenza A virus, including 19 H1N1, a single H1N2 and 9 H3N2 strains were obtained. Genetic characterization of these viruses was reported in a previous study
. Swab sample transport medium contained minimum essential medium (MEM), 2 × 107 IU/L penicillin G, 1 × 107 IU/L streptomycin, 100 mg/L gentamicin, 100 mg/L nystatin, 100 mg/L polymyxin B and 1000 mg/L sulfanilamide. SIV-positive samples suspected of containing bacteria (assessed by egg death and turbidity of the allantoic fluid) were used for bacterial isolation and identification.
Bacterial isolation and identification
SIV-positive swab samples suspected of containing bacteria were streaked onto LB agar plates and incubated at 37°C for 24 h. Bacterial colonies were collected and identified using a RiboPrinter Microbial Characterization System (DuPont Qualicon, DE, USA)
. Biochemical characterization was performed using an API 20 NE (BioMerieux, France) and analyzed with Apiweb software (BioMerieux, France) according the manufacturer’s instructions
. To confirm the identified result, several colonies selected randomly were used as template for 16S rRNA sequencing as previously described
. Sequences were analyzed by Basic Local Alignment Search Tool (BLAST) for species identification as previously described
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by disc diffusion methods recommended by the Clinical and Laboratory Standards Institute (CLSI). The antimicrobial discs tested included ofloxacin, levofloxacin, streptomycin, sulfadimidine, gentamicin, azithromycin, trimethoprim, ciprofloxacin, minocycline, ampicillin, amoxicillin, novobiocin, vancomycin, nitrofurantoin, cefotaxime, ceftazidime. (supplied by Tiantan Company of Pharmaceutical and Biological Products Development, Beijing, China). Determination of the antimicrobial susceptibility test was performed according to the manufacturer’s instructions, which followed the criteria of the CLSI. Reference strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control organisms in all antimicrobial susceptibility tests.
To investigate the interaction of SIV and S. maltophilia in influencing viral susceptibility and transmissibility in mammalian hosts, SPF/VAF (virus antibody free) Hartley strain female guinea pigs weighing 300–350 g and serologically negative for influenza virus were used. Animal experiments were approved by the Beijing Association for Science and Technology, the approve ID is SYXK (Beijing) 2007–0023, and complied with the guidelines of Beijing laboratory animal welfare and ethical of Beijing Administration Committee of Laboratory Animals. Zoletil 100 (tiletamine-zolazepam; Virbac S.A., Garros, France) was used to anesthetize animals by intramuscular injection (10–15 mg/kg).
Susceptibility in guinea pigs
Animals were inoculated with SIV or S. maltophilia alone or by co-infection of SIV and S. maltophilia. Groups of 18 animals were anesthetized and inoculated intranasally with 100 μL (106 50% egg infection dose, EID50) of virus, or 100 μL of 1.5 × 107 colony forming units (CFU) S. maltophilia, or a 100 μL mixture of the virus (106 EID50) and bacteria (1.5 × 107 CFU). Animals inoculated with Dulbecco’s phosphate-buffered saline (DPBS) were used as controls. On days 1, 2, 4, 6 and 8 p.i., three animals from each group were euthanized. Nasal washes, tracheas and lungs were collected and a part of each organ was homogenized in DPBS-A (DPBS containing 2,000 U/mL penicillin G and 2.5 mg/mL streptomycin) and then 0.10 mg/mL vancomycin was added (the S. maltophilia isolate was sensitive to vancomycin) for virus titration by EID50 assay. Nasal washes were performed by instilling a total of 1 mL of DPBS-A into the nostrils and collecting liquid runoff into a sterile Petri dish. To confirm successful bacterial inoculation, tracheas and lungs were also collected for isolation and identification on day 4 p.i., as previously described
. Groups of the remaining three animals were observed for 2 weeks for body weight, signs of disease, and tested for SIV seroconversion on day 14 p.i.
Transmissibility in guinea pigs
For the contact transmission study, groups of 15 naive guinea pigs were housed with the virus-inoculated, or virus and bacteria co-inoculated animals at 24 h p.i. and observed for 21 days. On days 2, 4, 6 and 8 p.i., the nasal washes, tracheas and lungs of three contact animals for each group were collected and titrated for EID50 assay. Groups of the remaining three animals were observed over 21 days for body weight and signs of disease, and tested for seroconversion on day 21 post-contact.
On day 4 p.i., trachea and lung specimens from inoculated guinea pigs were fixed in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Sections (4-μm thickness) were stained with hematoxylin and eosin (HE).
Differences in viral titers between single virus-inoculated and virus-bacterial co-inoculated groups were compared using Student’s t-test. A P-value less than 0.05 was considered statistically significant.