Lung involvement
Prior to inoculation all calves appeared clinically normal. Pasteurella multocida was isolated from pharyngeal swab samples from 34 of 35 calves. M. haemolytica was isolated from 8 of 35 calves. M. haemolytica positive pharyngeal swab calves were spread across all five treatment groups (1/4 in control group, 1/8 in leukotoxin negative B. trehalosi group, 2/8 in leukotoxin positive B. trehalosi group, 1/7 in M. haemolytica group, 3/8 in leukotoxin negative B. trehalosi and M. haemolytica combination group).
Six of the 35 enrolled calves were euthanized prior to day 9 of the study. Three calves (3/7) from the M. haemolytica group (days 2, 3 and 3) and three calves (3/8) from the leukotoxin negative B. trehalosi and M. haemolytica combination treatment (days 3, 5 and 8) were euthanized according to protocol because of a clinical assessment of moribund.
The mean estimated percent lung involvement was highest for the M. haemolytica group (49%). The mixed infection group of leukotoxin negative B. trehalosi and M. haemolytica had a mean lung involvement of 26%. The leukotoxin positive B. trehalosi had a mean lung involvement of 18% while the leukotoxin negative B. trehalosi mean lung involvement was estimated to be 13%. The control group had a mean lung involvement estimate of 13%. There was evidence of a statistically significant (p = 0.018) difference for mean percent total lung involvement between the M. haemolytica group and the leukotoxin negative B. trehalosi group. There were no significant differences between the other treatment groups. Even though the control group had a lower mean percent lung involvement than the leukotoxin negative B. trehalosi treatment group, the low number (4 calves) of study subjects in the control group did not have sufficient power to make it significantly different from the M. haemolytica treatment group. The mean and median values were similar in all treatment groups except the mixed infection group. This group had a high amount of variability within it as evidenced by a mean percent lung involvement of 26% and a median percent lung involvement of 5%. The high variability made drawing conclusions difficult in this group (Figure 1).
Free Hp
Other studies have found that serum Hp is nearly undetectable in healthy cattle [8]. Concentrations up to about 200 μg/mL are considered acceptable for healthy animals [2],[7],[9],[16]. Concentrations between about 200 and 400 μg/mL are associated with mild inflammation and concentrations greater than about 400 μg/mL suggest severe inflammation [7]. Serum Hp concentration does not differ with age or sex in cattle [2],[7],[9].
In this study, 11 calves (2/4 in the control group, 2/8 in the leukotoxin negative B. trehalosi group, 4/8 in the leukotoxin positive B. trehalosi group, 1/7 in the M. haemolytica group, and 2/8 in the leukotoxin negative B. trehalosi and M. haemolytica combination group) had serum Hp concentrations greater than 200 μg/mL prior to inoculation. Two of the calves had low serum concentrations of Hp-MMP 9 indicating the presence of chronic inflammation. M. haemolytica was cultured from the pharyngeal swabs of both calves. Nine of the calves with high serum concentrations of Hp also had high serum concentrations of Hp-MMP 9 indicating the presence of chronic inflammation which was associated with an acute inflammatory event. Three of these calves were culture positive for M. haemolytica from pharyngeal swabs. Nineteen calves had no detectible serum Hp prior to inoculation.
The interaction between treatment group and bleeding date was a statistically significant effect (p = 0.01). The mean serum Hp concentration for the leukotoxin positive B. trehalosi treatment group was significantly (p = 0.008) different from the other treatment groups for the pre-inoculation bleeding date, indicating that more calves with evidence of chronic inflammation prior to commencement of the study were randomly assigned to this treatment group. On the fourth and sixth bleeding dates (days 7 and 10) there was a significant (p < 0.05) difference in the mean Hp concentration between the M. haemolytica treatment group and the leukotoxin negative B. trehalosi treatment group and the leukotoxin negative B. trehalosi and M. haemolytica combination group. The M. haemolytica treatment group demonstrated significant elevations in serum Hp concentrations from the first two bleeding dates to the third bleeding date (day 5) (p = 0.02). The elevation in serum Hp concentration remained significant (p < 0.02) throughout the study. This finding is in agreement with other studies that have demonstrated an increase in serum Hp after M. haemolytica infection [4],[6]. Concentrations in this study peaked on day 7 rather than day 3 as previously reported. A decrease in the serum Hp concentration for the mixed infection group can be observed between the second and third bleeding dates (days 3 and 5). This observation supports findings by Dassanayake et al. suggesting that B. trehalosi is capable of inhibiting the growth of M. haemolytica in vivo [17]. In this study the difference in serum Hp concentration is not significantly different from the other treatment groups. Two calves from this treatment group were euthanized before the third bleeding time point. The smaller treatment group likely did not have enough power for a statistical difference to be observed (Figure 2).
Hp-MMP 9
The mean serum Hp-MMP 9 concentration for all treatment groups prior to inoculation was 104 ng/mL, with a minimum value of 0 ng/mL, a maximum value of 752.2 ng/mL, and a median value of 4.15 ng/mL. Little to no Hp-MMP 9 should be detectible in the serum of healthy calves [6]. Sixteen calves had no detectible serum Hp-MMP 9 concentration prior to inoculation. For this study a serum concentration greater than 20 ng/mL was considered high. Thirteen calves had high serum Hp-MMP 9 concentrations prior to inoculation. Four of the thirteen calves had low serum concentrations of Hp, suggesting the presence of a mild acute inflammatory process. None of these 4 calves were culture positive for M. haemolytica from the pharyngeal swabs. Nine of the thirteen calves had high serum concentrations of Hp and were discussed previously.
Treatment group, bleeding date, and their interaction were all significant (p < 0.01) effects. On the fourth bleeding date (day 7) the mean serum Hp-MMP 9 concentration for the M. haemolytica group became significantly different from the negative control, the leukotoxin negative B. trehalosi, and the leukotoxin negative B. trehalosi and M. haemolytica combination groups (p = 0.01, p = 0.009, and p = 0.004 respectively). This difference continued throughout the study. On the fifth bleeding date (day 9) the mean serum Hp-MMP 9 concentration for the M. haemolytica group became significantly different from the leukotoxin positive B. trehalosi group (p = 0.006) and remained different throughout the rest of the study. The M. haemolytica group demonstrated significant elevations in serum Hp-MMP 9 concentrations from the first three bleeding dates to the last three bleeding dates (p < 0.02). The leukotoxin positive B. trehalosi treatment group demonstrated a small but significant (p < 0.004) elevation in serum Hp-MMP 9 concentrations from the pre-inoculation bleeding date to the second bleeding date (day 3) that continued throughout the study (Figure 3). This finding is consistent with other studies that have demonstrated the rapid recruitment and accumulation of neutrophils at the onset of BRD [6] as well as the action of leukotoxins to stimulate the active degranulation of bovine neutrophils [5].
Correlations
There was statistically significant (p < 0.0001) evidence of a strong (r = 0.7) correlation between Hp and Hp-MMP 9 serum concentrations as both ranked and absolute values prior to inoculation. Evidence of a relationship remained statistically significant over time (p < 0.0001), but the strength of the relationship varied depending on if a Pearson (absolute values) or Spearman (ranked data) correlation coefficient was calculated. The Pearson correlation coefficient was r = 0.63; whereas, the Spearman correlation coefficient was r = 0.79. This strong relationship was expected since both substances have been established as biomarkers for inflammation and the test for free Hp measures Hp-MMP 9 contribution also.
There was statistically significant evidence (p < 0.05) of a moderate correlation (r = 0.44 and r = 0.35) that became stronger over time (r = 0.67 and r = 0.48) between Hp-MMP 9 and percent lung involvement for both ranked and absolute values respectively. There was statistically significant evidence (p = 0.04) of a moderate (r = 0.35) correlation between free Hp and percent lung involvement for ranked, but not absolute values prior to inoculation. This correlation became stronger (r = 0.54, p = 0.0008) when values were averaged over the time of the study. The correlation between free Hp and percent lung involvement for absolute data was also statistically significant when averaged over time (r = 0.41, p = 0.015).
This correlation data indicates that Hp-MMP 9 serum concentrations may be a good ante-mortem diagnostic indicator of lung damage that is found at the time of the calf’s death. This data also supports the conclusion from other studies that Hp-MMP 9 serum concentration is a better diagnostic test for lung damage than free Hp serum concentration [1]. Future studies should be conducted to determine reference ranges of serum Hp-MMP 9 for different levels of disease severity.