Animals that met the inclusion criteria aged 1 week to 7 months. In total, 13 nBAL samples were eligable, of which the initial culture results at T0 showed 6 dominant cultures with 1 clinically relevant pathogen (4 M. haemolytica, 2 P. multocida) and 7 mixed cultures with both M. haemolytica and P. multocida (3 pure cultures, meaning the presence of 1 bacterial species on the agar plate with more than 2 colonies, containing only M. haemolytica and P. multocida and 4 dominant cultures with also some contaminants present). In total, M. haemolytica was isolated from 11 samples (84.6%) with an average log concentration of 4.0 at T0, and P. multocida from 9 samples (69.2%) with an average log concentration of 3.7 at T0. Trueperella pyogenes was isolated from one sample and Moraxella bovis from two samples at T0, both in mixed cultures. H. somni was not isolated. Contaminants present in the dominant cultures were Streptococcus spp., Staphylococcus spp., Bacillus spp., Escherichia coli and Rothia nasimurium with an average log concentration of 2.8 at T0.
The effect of the various temperatures and storage periods after sampling on the number of positive samples for M. haemolytica is presented in Figure 1. In general, the number of samples from which M. haemolytica could be isolated, decreased gradually over time. The higher the storage temperature, the earlier the number of positive samples started to decrease, i.e. at 2 hours of storage at 36°C, at 24 hours of storage at 23°C and at 48 hours of storage at 0°C and 8°C. During storage at 0°C and 8°C, the number of positive samples remained stable up to 24 hours after sampling, varying between 10/11 and 11/11 in this period, whereas at 23°C, only 7/11 samples remained positive after 24 hours of storage. When samples were stored at 36°C for 48 hours, M. haemolytica could be isolated from only 1 sample, while this was 4/11 for 23°C and 8/11 for both 0°C and 8°C (Figure 1). Significance was only reached after 6 hours of storage at 36°C compared to lower temperatures.
An overall slight decrease in M. haemolytica concentration occurred over time (Figure 2). At a storage temperature of 36°C, the average log concentration of M. haemolytica decreased after 2 hours of storage from 4 to 3.5 and remained stable until 48 hours after sampling. No statistical significant difference was seen between the different temperatures and the time after sampling.
The effect of storage temperature and time on the number of samples from which P. multocida could be isolated is presented in Figure 3. A significant difference of isolation is only seen at 48 hours of storage at a temperature of 0°C or 8°C, 23°C and 36°C. A decline in the number of positive samples from which P. multocida was isolated was observed throughout the experiment for storage at 36°C, where only 4/9 positive samples were retrieved at 24 hours of storage and no positive samples could be retrieved at 48 hours (Figure 3). This decline in the number of positive samples was due to both contaminant overgrowth and a decreased viability. This decreased viability of P. multocida was noticed at a storage temperature of 36°C starting from 24 hours after sampling and at a storage temperature of 23°C starting from 48 hours after sampling. When samples were maintained for 48 hours at 23°C, only 3/9 samples were found positive. At a storage temperature of 0°C or 8°C, the number of positive samples remained stable until 24 hours after sampling, with an isolation rate of 8/9 to 9/9. At 48 hours of storage at a temperature of 0°C, 1 initially P. multocida positive sample was negative due to decreased viability. When stored at 8°C for 48 hours, 2 initially P. multocida positive samples were negative, one due to decreased viability and one due to contaminant overgrowth.
Compared with M. haemolytica, the average concentration of P. multocida remained more stable until 24 hours of storage at a temperature of 0°C, 8°C and 23°C (Figure 4). At a storage temperature of 36°C, the P. multocida concentration slightly decreased starting from 6 hours after sampling, with a concentration below detection limit at 48 hours after sampling (Figure 4). Accordingly, no positive samples of P. multocida were retrieved after storage at 36°C for 48 hours, even in the absence of contamination overgrowth. No statistical significant difference was seen between the different temperatures and the time after sampling.
From the 13 samples collected, 10 samples contained microbial contamination at T0. Of the 3 initial samples that were not contaminated at T0, 1 sample showed microbial contamination starting from 2 hours after sampling at a temperature of 36°C. The other two initially negative samples showed sporadically microbial contamination with a concentration close to the detection limit. Results of the influence of bacterial contamination on the isolation rate of M. haemolytica and P. multocida in the clinical nBAL samples are presented in Table 1. A statistically significant negative association was seen between the presence of contaminants and the presence of P. multocida. An odds ratio of 0.32 was obtained for P. multocida (P=0.04), meaning that the presence of contaminants reduced the odds of isolating P. multocida. In contrast, for M. haemolytica no significant effect of the presence of contaminants on isolation rates could be evidenced (P=0.70). An average initial contaminants log concentration of 2.8 CFU/mL was observed (Figure 5). This concentration remained stable during the first 8 hours of storage, regardless of storage temperature. However, after 24 hours of storage at 36°C, the average contaminants log concentration increased to 4.4. After 48 hours of storage, an average contaminants log concentration of 4.7 and 5.1 was reached for storage at 23°C and 36°C, respectively (Figure 5).
Table 1: The effect of the presence of microbial contamination with a concentration of ≥ 1x102 CFU/mL of each sample on the isolation of M. haemolytica and P. multocida
Contaminants
|
Odds ratio
|
Confidence Interval
|
P-value
|
|
Negative
|
Positive
|
Mannheimia haemolytica
|
Negative
|
37.4%
|
62.2%
|
0.70
|
0.58 -1.41
|
0.70
|
Positive
|
45.8%
|
54.2%
|
Pasteurella multocida
|
Negative
|
21.2%
|
78.8%
|
0.32
|
0.37 -0.83
|
0.04
|
Positive
|
45.7%
|
54.3%
|