Animals and experimental design
The animals were born on a dairy farm and were transported to the University of São Paulo on the first day of life after receiving the main neonatal care and ingesting colostrum corresponding to at least 10% of their live weight along with a consent document from the owner for use of the animals in the study.
After arriving at the university, to ensure the successful transfer of passive immunity, the total serum protein should be greater than 5.0 g/dL [42] and glutaraldehyde agglutination test faster than five minutes [43]. Furthermore, a complete blood count was performed, and they should be within normal ranges for their respective age and specie [44]. In the meantime, all animals were also subjected to physical examination [45], which include the following parameters: animal’s general condition, respiratory and heart rates, body temperature, intestinal motility rate, degree of body hydration, apparent color of mucous and palpation of lymph nodes. If any alteration was found, the animal was excluded from the experiment.
Sixteen male Holstein dairy calves with median weight of 31,5 kg (range, 23 to 40 kg) were all housed in individual suspended iron cages that included a shelter and outdoor access. They were feeded milk and calf feed twice a day until the day 30 and after this day tifton hay and ad libitum access to water.
Calves were randomized assigned to three groups on the day of birth (day 0). Calves in group A (n = 6) were inatranasally inoculated as previously proposed [11, 18, 46] with 1 mL of a polyvalent vaccine on days 15 and 36, calves in group B (n = 5) were inatranasally inoculated on days 45 and 66, and calves in group C (n = 5) were not inoculated and served as a control group. Bronchoalveolar lavage (BAL) samples were obtained by endoscopy [20] from calves in groups A and C on days 15, 36 and 57 and from calves in groups B and C on days 45, 66 and 87.
Mucosal immunization was implemented because it was shown to be effective in calves and had less interference from maternal antibodies [18, 47, 48]. The commercial vaccine (CattleMaster Gold FP 5/L5, Pfizer Animal Health, Montreal, Canada) used contains a lyophilized preparation of modified live strains of IBR, PI3, and BRSV, inactivated BVDV types 1 and 2 and inactivated cultures of five Leptospira sp. serovars in a liquid preparation together with an adjuvant Quil-A.
After the study, the animals were donated to a dairy farmer, who kept the animals in the production system.
Clinical examination
Firstly, the respiratory and heart rates, body temperature, rumen motility rate, degree of body hydration, apparent color of mucous and palpation of lymph nodes were performed. Then, the calves were subjected to specific examination of the respiratory system by the direct inspection, palpation, percussion, and auscultation. The presence of secretion in the respiratory system was assessed by endoscopy using a flexible video gastroscope.
Bronchoalveolar lavage sampling
The animals were sedated with 0.02 mg/kg of xylazine (Rompun 2%, Bayer Animal Health) and after a few minutes were placed in a lateral recumbent position, and then approximately 50 mL of BAL was recovered by endoscopy. To maintain cell viability, all of the samples were kept on ice until processing, which did not exceed a maximum of 4 h after collection. The samples were subjected to two washes with phosphate buffered saline, centrifuged and resuspended to a concentration of 2 × 106 viable cells mL− 1, as previously described [20].
Identification of neutrophils, monocyte-like macrophages and vacuolized macrophages
The neutrophils, monocyte-like macrophages and vacuolized macrophages were identified by flow cytometry (FACSCalibur™, Becton Dickinson Immunocytometry System™, San Diego, EUA) using the procedure proposed by Sladek and Rysanek [49] with some modifications. Briefly, the region distribution of BAL cells in dot plots was differentiated by their forward scatter and side scatter parameters into neutrophils, monocyte-like macrophages and vacuolized macrophages (Fig. 1A). After identifying each leukocyte population, the neutrophils (CD14− cells; Fig. 1B), vacuolized macrophages (CD14+ cells; Fig. 1C) and monocyte-like macrophages (CD14+ cells; Fig. 1D) were analyzed based on their cytoplasmic granularity and mean fluorescence intensity following a two-step fluorescent immunolabeling protocol using primary anti-bovine monoclonal antibody specific for CD14 cells (cat. no. MM61A, VMRD Pullman Inc. Corp, Pullman, USA) and secondary goat anti-mouse IgG1 conjugated to phycoerythrin-Cy5 (cat. no. M32018, Life Technologies, San Diego, USA) [20]. An unstained control, secondary antibody control and single stained samples were also prepared as compensation controls. FlowJo software® (TreeStar Inc., Ashland, OR, USA) was used to analyze the data.
Intracellular reactive oxygen species production
Reactive oxygen species production by alveolar neutrophils, monocyte-like macrophages and vacuolized macrophages was evaluated by flow cytometry using 2',7'-dichlorofluorescein diacetate (cat. n. D6883, Sigma Aldrich, St. Louis, MO, USA) as a probe [50–54]. For this assay, 10,000 to 15,000 BAL cells were examined per sample. An unstained control and single stained samples were also prepared as compensation controls. FlowJo software® (TreeStar Inc., Ashland, OR, USA) was used to analyze the data.
Phagocytosis assay
The phagocytosis assay of alveolar neutrophils, monocyte-like macrophages and vacuolized macrophages was performed by flow cytometry using propidium iodide-labeled Staphylococcus aureus as previously described [20, 50–53] at multiplicity of infection of 120. For this assay, 10,000 to 15,000 BAL cells were examined per sample. An unstained control and single stained samples were also prepared as compensation controls. FlowJo software® (TreeStar Inc., Ashland, OR, USA) was used to analyze the data.
Statistical analysis
Firstly, the results of the flow cytometry were corrected for autofluorescence by substraction, which was defined as the fluorescence that was associated with unlabeled freshly cells from the same calf. The statistical analysis was performed using an especific software (GraphPad Software, Inc., San Diego, CA, USA). First, the Gaussian distribution was verified by the Kolmogorov-Smirnov test. To compare the differences between groups (vaccinated vs unvaccinated), Student’s t-test for unpaired data was used for the data with Gaussian distributions, and the Mann-Whitney test was used for the non-parametric data. To evaluate differences among the days in each group, a repeated measures ANOVA and the post hoc Newman-Keuls test were used for the data with a normal distribution. The Kruskal-Wallis test and Dunn’s post hoc test were applied to the non-parametric data. Significance was set at P < 0.05 unless indicated otherwise.