Acute phase proteins as local biomarkers of respiratory infection in calves

Abstract

Background: Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci.

Results: Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP-and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]--and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection.

Conclusion: In conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.

Fig. 1

Kinetics of respiratory score and lipopolysaccharide-binding protein (LBP) in blood serum of calves. (a) Respiratory score and (b) concentration of LBP in blood serum of calves before and after inoculation with Chlamydia (C.) psittaci. Blue bars represent time-points of broncho-alveolar lavage and corresponding blood sampling. Blue panel with solid line: exact time point, blue panel with dashed line: blood collection one day after broncho-alveolar lavage. Post inoculation values were compared to the value 1 h before inoculation (−1 h) with the Wilcoxon signed rank test with Holm adjustment. -7 d: 7 days before inoculation; −4 d: 4 days before inoculation; dpi: days past inoculation; grey: animals prior to inoculation with C. psittaci; white: animals after inoculation with C. psittaci (n = 13). The 13 calves served as untreated controls in two previous treatment studies where the general clinical scores and concentrations of LBP in blood sera have been published separately [31, 34]

Fig. 2

Bovine serum albumin (BSA) in calves. (a) BSA in blood sera of calves before and after inoculation with Chlamydia (C.) psittaci. Post inoculation blood values were compared to the value 1 h before inoculation (−1 h) with the Wilcoxon signed rank test with Holm adjustment. -7 d: 7 days before inoculation; −4 d: 4 days before inoculation; dpi: days past inoculation; grey: animals prior to inoculation with C. psittaci; white: animals after inoculation with C. psittaci (n = 13). (b) BSA in BALF supernatant of healthy and C. psittaci-infected calves. BALF values of infected animals were compared to the first sample from healthy controls, since at that time point the age difference between the animals was lowest (Mann–Whitney-U test with Holm adjustment). 1, 2, 3: first, second, and third sample from healthy controls; light blue: healthy control animals (n = 6); white: animals after inoculation with C. psittaci (n = 13). #: 0.05 ≤ P < 0.1; *: 0.01 ≤ P < 0.05

Fig. 3

Relationships between clinical signs and markers of acute phase reaction assessed in blood (a) or BALF (b) in calves with an acute respiratory infection induced by Chlamydia psittaci. Models of multiple regressions (n = 39). a Dependent variable: General Clinical Score (z-axis). Independent variables: concentrations of bovine serum albumin (BSA; x-axis) and LBP (y-axis) in blood serum. R² = 35.4 %. P-Value of the model = 0.0004. b Dependent variable: Respiratory Score (z-axis). Independent variables: concentrations of Lipopolysaccharide Binding Protein (LBP; x-axis) and Haptoglobin (Hp; y-axis) in broncho-alveolar lavage fluid (BALF). R² = 55.3 %. P-Value of the model = 0.0000

Fig. 4

Protein fractions in blood sera of calves before and after inoculation with Chlamydia psittaci. Post inoculation values were compared to the value 1 h before inoculation (−1 h) with the Wilcoxon signed rank test with Holm adjustment. -7 d: 7 days before inoculation; −4 d: 4 days before inoculation; dpi: days post inoculation; grey: animals prior to inoculation with Chlamydia (C.) psittaci; white: animals after inoculation with C. psittaci (n = 13); #: 0.05 ≤ P < 0.1; *: 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01

Fig. 5

Quotients between [local marker]/[BSA] in BALF supernatant of healthy and Chlamydia psittaci infected calves. Values of infected animals were compared to the first sample from healthy controls (Mann–Whitney-U test with Holm adjustment). Lf: lactoferrin; BSA: bovine serum albumin; CRP: C-reactive protein; LBP: lipopolysaccharide binding protein; Hp: haptoglobin; dpi: days after inoculation; 1, 2, 3: first, second, and third sample from healthy controls; white: animals after inoculation with C. psittaci (n = 13); light blue: healthy control animals (n = 6); #: 0.05 ≤ P < 0.1; *: 0.01 ≤ P < 0.05; ** 0.001 ≤ P < 0.01; *** P < 0.0001