In vitro evaluation of the effect of transport medium, temperature, and time on the recovery of Mannheimia haemolytica and Pasteurella multocida

Appropriate sample collection, storage conditions, and time for transport to the laboratory are important for an accurate diagnostic result. We evaluated the effects of transport storage medium type, time of storage, and storage temperatures on Mannheimia haemolytica (MH) and Pasteurella multocida (PM) recovery using an in vitro model simulation. A quantitative culture method, using colony-forming units per milliliter, was used to recover MH or PM by an in vitro model with cotton swabs. Three independent trials were conducted, in which cotton swabs were inoculated with MH or PM and placed in either (1) a sterile 15-mL polypropylene tube without transport medium (dry), (2) Amies culture medium with charcoal (ACM), or (3) Cary-Blair transport agar (CBA). Swabs were evaluated for recovery of MH or PM when stored at 3 temperatures (4°C, 23°C, or 36°C) and after storage for 8 h, 24 h, or 48 h. From all study group combinations, a total of 162 individual independent swabs were evaluated. The nonparametric Dunn all-pairs approach was used to compare the proportion of culturable bacteria, between the various storage media, temperature, and time point combinations. The proportion of MH in samples stored at 4°C was significantly higher for ACM and CBA than dry storage at 24 and 48 h. The MH samples stored at 36°C had a significantly higher proportion for ACM and CBA than dry storage at 24 h. The proportion of PM in samples stored at 4°C was significantly lower for ACM compared with dry at 8 h but significantly higher at 48 h. The PM samples stored at 23°C in ACM had a significantly higher proportion than dry samples at 24 h, and, at 48 h, ACM and CBA had a significantly higher proportion than the dry group. All swabs stored at 36°C for 48 h had a proportion close to zero, indicating decreasing diagnostic efficacy. These results support the use of transport media such as ACM and CBA for increasing the detection of PM and MH from samples, especially when samples are exposed to high temperatures. The combination of longer periods from collection of samples to diagnostic evaluation (>24 h) and higher storage temperatures (>23°C) were shown to significantly impair diagnostic accuracy.