Serological results showed a similar response pattern for naturally and experimentally infected cattle, with a few relevant variations. In both naturally and experimentally infected cattle, globulins, albumin and AChE showed significant variations. Haptoglobin was one of the most relevant markers in experimentally infected cattle, whereas ADA showed significant variations in naturally infected cattle. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during chronic infection. The levels of each biomarker at 0 dpi in experimentally infected calves and in non-infected field cattle were very similar except for PON-1, which was lower in field animals than in experimentally infected calves.
Acute-phase and oxidative stress responses in experimentally infected calves
The serological results of the different markers are shown in Fig. 1.
Haptoglobin concentrations were increased from 8 to 12 days post-infection (dpi) in all infected groups. This increase was significant in the subcutaneously inoculated group at 8 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.88, df = 144, P < 0.001) and 12 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 10.02, df = 144, P < 0.0001), and concentrations decreased thereafter. In both the intravenously and intradermally inoculated groups, this increase was not significant at 8 dpi, and then the levels decreased, followed by a significant increase at 22 dpi in the intravenously inoculated group (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 4.318, df = 144, P < 0.05) and at 29 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 4.55, df = 144, P < 0.01) and 33 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.82, df = 144, P < 0.001) in the intradermally inoculated group. Next, Hp levels decreased until the end of the study.
Total protein levels were similar among the three inoculated groups with the exception of a few relevant findings when compared to the control group: higher levels at 54 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 3.83, df = 144, P < 0.05) in the subcutaneously inoculated group; higher levels at days 33, 47, 54 and 75 dpi (ANOVA, F(15, 135) = 5.94, P < 0.05) in the intravenously inoculated group; and a significant decrease at 22 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 5.05, df = 144, P < 0.001), followed by an increase at 33 dpi (ANOVA followed by Tukey’s post hoc multiple comparison tests, q = 3.13, df = 144, P < 0.05) and maintenance of higher levels at 47 and 75 dpi (ANOVA, F(15, 135) = 5.94, P < 0.05) in the intradermally inoculated group.
Globulin levels showed a significant increase compared to the control group from 22 dpi in the subcutaneously inoculated group (ANOVA, F(15, 135) = 7.66, P < 0.05) and from 29 dpi in the intravenously (ANOVA, F(15, 135) = 7.66, P < 0.01) and intradermally (ANOVA, F(15, 135) = 7.66, P < 0.01) inoculated groups and thereafter.
AChE values increased from 25 dpi. In general terms, in all infected groups, the levels were similar to or lower than those detected in the control group. The most relevant differences corresponded to the subcutaneously and intravenously infected group at 47 dpi, showing lower levels than the control group (ANOVA, F(15, 135) = 15.78, P < 0.05) until the end of the study, and in the intravenously infected group at 64 dpi (ANOVA, F(15, 135) = 15.78, P < 0.01) and 75 dpi (ANOVA, F(15, 135) = 15.78, P < 0.0001). The values increased similarly in both the intradermally infected group and the non-infected group.
The levels of albumin were lower in all inoculated groups than in the control group. This decrease was significant in the subcutaneously (ANOVA, F(15, 135) = 6.53, P < 0.0001) and intravenously (ANOVA, F(15, 135) = 6.53, P < 0.01) inoculated groups from 8 dpi onwards and in the intradermally inoculated group from 12 dpi onwards (ANOVA, F(15, 135) = 6.53, P < 0.01).
Significantly lower PON-1 levels were observed at 15 (ANOVA, F(15, 135) = 7.25, P < 0.01) and 19 dpi (ANOVA, F(15, 135) = 7.25, P < 0.01) in the subcutaneously infected group and at 22, 26, 29 and 33 dpi (ANOVA, F(15, 135) = 7.25, P < 0.05) in the intradermally infected group compared to the control group.
ADA levels were generally higher in all infected groups. The most relevant differences corresponded to 19 and 33 dpi in the subcutaneously infected group (ANOVA, F(15, 135) = 3.33, P < 0.05) and to 75 dpi in the intradermally infected group (ANOVA, F(15, 135) = 3.33, P < 0.05).
Acute-phase and oxidative stress responses in naturally infected cattle
The serological results of the different markers are shown in Fig. 2. Similar results were obtained regardless of the criteria employed to classify the animals in the different categories (panel a: sterile acutely and chronically infected bulls versus fertile subclinically infected bulls; panel b: bulls with acute, chronic or subclinical infection based on clinical signs and serological results). However, differences were more evident in panel b when animals were classified according to serological results and clinical signs compared to a more restrictive criterion where only sterile and fertile bulls were considered.
Non-significant variations in Hp, AChE and PON-1 values were observed among the groups studied (Fig. 2). The remaining markers showed significant differences between some of the groups, as mentioned below.
Total protein levels were higher in chronically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 28.66, df = 2, P < 0.05) and acutely infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 15.51, df = 2, P < 0.05) as shown in Fig. 2a. These values were similar in those groups with clinical signs (Fig. 2b) in which levels of total protein were higher in chronically infected and subclinically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 32.93, df = 2, P < 0.05 and χ2 = 18.37, df = 2, P < 0.07, respectively) and early acutely infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 51.28, df = 2, P < 0.001 and χ2 = 36.72, df = 2, P < 0.01, respectively).
Globulins levels were significantly higher in chronically and subclinically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 32.78, df = 2, P < 0.01 and χ2 = 19.26. df = 2, P < 0.01, respectively) (Fig. 2a). The similar is found in bulls with clinical signs (Fig. 2b), where significantly higher levels in were chronically and subclinically infected bulls than in non-infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 38.15, df = 2, P < 0.01 and χ2 = 22.44, df = 2, P < 0.05, respectively) and early acutely infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 48.12, df = 2, P < 0.01 and χ2 = 32.40, df = 2, P < 0.05, respectively).
Higher values of AChE but not significant were observed in late acute and chronic infections (Fig. 2).
Albumin values were lower in all infected groups than in the non-infected group. These differences were significant in acutely infected bulls [acute infection: Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 45.53, df = 2, P < 0.05 (Fig. 2a); early acute infection: χ2 = 4.15, df = 2, P < 0.001 (Fig. 2b); late acute infection: χ2 = 45.43, df = 2, P < 0.01 (Fig. 2b)] and in subclinically infected bulls (χ2 = 17.17, df = 2, P < 0.05 in Fig. 2a, and χ2 = 18.79, df = 2, P < 0.06 in Fig. 2b) compared to non-infected bulls.
Non-significant variations in PON-1 values were observed among the groups studied (Fig. 2a, b). The highest levels corresponded to bulls with chronic infection, followed by bulls with acute infection and subclinically infected bulls (Fig. 2a). The lowest levels corresponded to bulls with early acute infection (Fig. 2b).
Finally, the highest ADA levels corresponded to bulls with an early acute infection, followed by bulls with a late acute infection, chronically infected bulls and finally subclinically infected cattle. Significant differences were observed between bulls with an early acute infection and subclinically infected bulls (Kruskal–Wallis H-test followed by Dunn’s multiple comparison test: χ2 = 29.55, df = 2, P < 0.05) and the negative control (χ2 = 33.66, df = 2, P < 0.01).