Abstract
B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12–17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies.
Highlights
Bacterial suspensions of each dose were sprayed through the device and collected into 900 μl of phosphate-buffered saline (PBS), serially diluted, and plated on tryptic soy agar (TSA) in duplicate to calculate the number of viable bacteria
This study proves that the device is a reliable means of generating an infectious aerosol, and passage through the MicroSprayer1 does not adversely affect the viability of the bacteria
We evaluated the ability of the device to inoculate guinea pigs with low doses (101, 102,103) or high doses (106, 107, 108) of B. melitensis 16M
Summary
This study was carried out in an approved facility in strict accordance with all university and federal regulations. All guinea pig experimentation was reviewed and approved by the Texas A&M University Laboratory Animal Care and Use Committee (protocol: 2015–0036). The protocol was approved and is in accordance with the Institutional Animal Care and Use Committee (IACUC) policies of Texas A&M University. Texas A&M is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Outbred Harley female guinea pigs (n = 44) weighing approximately 300–350 g were obtained from Charles River Laboratories and housed individually in microisolator caging in a biosafety level three facility. A modified Karnofsky performance status scoring system was used to evaluate the guinea pigs daily to determine if early removal from the study was required
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