Effects of Buprenorphine Treatment on Influenza Pathogenesis in the Ferret (Mustela putorius furo)

Abstract

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups ( n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

Figure 1.

Replication of IAV in buprenorphine-treated ferrets. Ferrets (group 1, buprenorphine on days 0 through 4; group 2, buprenorphine on days 5 through 9; group 3, saline controls) were inoculated intranasally with 106 pfu of Panama/99 IAV. Nasal wash specimens were collected on alternate days after inoculation and titered for the presence of infectious virus by using a standard plaque assay. Data given as mean (bar, 1 SD) values from 4 ferrets per group per day of specimen collection. *, P ≤ 0.05 by 2-way ANOVA. Limit of detection, 10 pfu/mL.

Figure 2.

Kinetic analysis of circulating lymphocytes during IAV infection in buprenorphine-treated ferrets. Ferrets (group 1, buprenorphine on days 0 through 4; group 2, buprenorphine on days 5 through 9; group 3, saline controls) were inoculated intranasally with 106 pfu of Panama/99 IAV. Blood was collected in EDTA vacuum phlebotomy tubes on the days indicated and analyzed on a hematology scanner. Mean average percentages of lymphocytes (LY), neutrophils (NE), basophils (BA), monocytes (MO), and eosinophils (EO) in whole blood are shown (means from n = 4 ferrets per group except for group 3, day 3 [n = 3 due to sample coagulation in one specimen prior to analysis]).

Figure 3.

Weight loss in IAV-infected ferrets receiving buprenorphine-treatment or saline. Ferrets were inoculated intranasally with 106 pfu of Panama/99 IAV. (A) Daily mean weight change from preinoculation body weight for all groups (n = 4 per group). (B) Ferrets received buprenorphine treatment (group 1, n = 4) or saline (group 3, n = 2) days 0–4 pi; (C) Ferrets received buprenorphine (group 2, n = 4) or saline (group 3, n = 2) on days 5 through 9. (B and C) Body weights were collected twice daily (0800 and 1600), with the span of buprenorphine or saline treatment shown in each graph; body weight percentages were set at 100% on the first day of buprenorphine administration.