# Effects of 3 Different Commercial Vaccines Formulations against BVDV and BHV-1 on the Inflammatory Response of Holstein Heifers

^{1}

^{2}

^{3}

^{*}

## Abstract

**:**

## 1. Introduction

^{+}lymphocytes (first interaction) in the lymph nodes, followed by signaling, results in the activation of the cell-mediated immune response. These lymphocytes subsequently differentiate into T helper 1 (Th1) or T helper 2 (Th2) effector T cells. The TCD8

^{+}lymphocytes and B-lymphocytes migrate from the thymus and bone marrow, respectively, to regional lymph nodes, where secondary immune interaction occurs between CD4

^{+}type-Th1, CD8

^{+}-cytotoxic T cells (CTL) or CD4

^{+}type-Th2, and B lymphocytes. After these interactions, TCD8

^{+}and B cells become effector or memory cells [12,14,15].

## 2. Materials and Methods

#### 2.1. Animals

#### 2.2. Vaccination and Management

^{®}, BD Diagnosis, Franklin Lakes, NJ, USA) into syringe of 5 mL (Platispack

^{®}, BD Diagnosis, Franklin Lakes, NJ, USA).The heifers received two doses of vaccines (5 mL), at a 21-day interval and unvaccinated group (control) received saline injection (5 mL) at the same 21day interval. The batch vaccines used for first and second dose: Vaccine A (Lot:001/15), Vaccine B (Lot:003/15), Vaccine C (Lot:002/15).

#### 2.3. Local and Systemic Reaction

^{2}).

#### 2.4. Blood Samples

^{®}, BD Diagnosis, Franklin Lakes, NJ, USA) into vacuum tubes (Vacutainer

^{®}, BD Diagnosis, Franklin Lakes, NJ, USA) with and without anticoagulant to obtain blood and serum, respectively. Blood samples were collected on study days at 0, 6, 24, 48, 72 and 168 h in a 2 mL polypropylene tube for assessing leukocyte and platelet counts (BD Vacutainer K2 Ethylenediaminetetraacetic acid - EDTA, 3.6 mg REF367841

^{®}; BD Diagnosis, Franklin Lakes, NJ, USA) and into two individual 10 mL glass tubes (BD Vacutainer Serum

^{®}; BD Diagnosis, Franklin Lakes, NJ, USA) for testing serum iron, Hp and antibody concentration.

#### 2.5. White Blood Count (WBC)

^{®}, Montpellier, France).

#### 2.6. Iron Concentration

#### 2.7. Haptoglobin

_{2}O

_{2}solution (0.02 mol/L) were added. After 10 min, absorbance at 490 nm was measured using a microplate reader. All samples were run in duplicate and the mean of each duplicate was used to calculate the final concentration based on the standard curve.

#### 2.8. Serum Virus Neutralization (SVN)

_{2}were done following a 1:10 (BVDV-1 NADL strain) or 1:2 (BHV-1-Los Angeles strain) dilutions using the cell culture medium MEM (minimum essential medium) containing 1% of Pen-Strep antibiotics and 5% BVDV Ab-free fetal bovine serum as the diluent. An amount of 50μL of serum in duplicates were added to wells of the plates, after which 50 μL of the respective virus solution containing TCID

_{50}/100 μL (50% tissue culture infective doses) was added. Plates were incubated for 18–24 h for BHV-1 and one hour for BVDV in a 37 °C oven with 5% CO

_{2}. After incubation the MDBK (Madin-Darby bovine kidney) cell suspension was added to each well of the plates. The serum neutralization tests for BVDV-1 Singer strain and BVDV-2 VS253 strain were done at the Department of Virology at University of Santa Maria using 100 to 200 infecting doses of each virus used. After this procedure the plate was incubated at 37 °C with 5% CO

_{2}for 4–5 days. Viral infectivity was indicated by the cytopathic effect (CPE) visible in the cell monolayer under inverted microscope. Antibody titer was expressed as the highest dilution of serum that completely inhibited the CPE in both wells of each dilution. Virus titer was determined by retitration [27].

#### 2.9. Statistical Analysis

^{®}version 9.4, SAS Institute, Cary, NC, USA). All variables were evaluated for Gaussian distribution by function guided data analysis. Some data presented no normal distribution and were subjected to a logarithmic transformation by log

_{10}, square root or inverse to obtain a normal distribution of the variables.

_{Tr}; Vaccines A, B, C and Control) and days (P

_{Day}; 0, 6, 24, 48, 72 and 168 h), as well as the interaction of treatment and day (P

_{TrxDay}; effects by a MIXED procedure (PROC-mixed, SAS), with least significant difference (LSD) post hoc test. The models were tested according to covariance structures, using Akaike information criterion (AIC). Differences were considered significant when p ≤ 0.05.

## 3. Results

#### 3.1. Local Vaccine Reactions

^{2}), compared to Vaccine A (27 mm

^{2}) and Vaccine C (30 mm

^{2}) after the first dose. In contrast, for the second dose of vaccine, there was an increase in skin thickness for Vaccine C (p = 0.0001; 61 mm

^{2}) compared to Vaccine A (34 mm

^{2}) and Vaccine B (44 mm

^{2}). The skin thickness of heifers control group was significantly less (ranged from 9 to 11mm

^{2}) than vaccinated heifers at 6h after the first dose and then at all times after the second dose.

#### 3.2. Systemic Vaccine Reactions

#### 3.3. White Blood Count (WBC)

^{3}/µL) had no treatment effect or Tr x day interaction after the first dose of the vaccine (p ≥ 0.050) (Figure 4a). On the other hand, the effect of day on the number of total leukocytes (p = 0.026) was detected, with a decrease in WBC in Vaccine B group between 6 h and 168 h. Regarding the second dose, only the treatment effect (p = 0.001) and the day (p = 0.0002) were observed. The maximum number of WBC was observed at 6 h in vaccinated and control groups, with a gradual decrease in subsequent moments. WBC number was higher in Vaccine C and lower in control group at 6 h and 168 h after the vaccination protocols.

^{3}/µL).

#### 3.4. Serum Iron Levels

#### 3.5. Haptoglobin Inflammation

#### 3.6. BVDV and BHV-1 Antibody Production

_{2}= 5.1) and vaccine C (GMT-log

_{2}= 5.1). Heifers receiving vaccine A seroconverted to BVDV-1 Singer strain (GMT log

_{2}= 0.1) at D21. At booster day (D42) group vaccine A had higher mean titers of Abs (GMT-log

_{2}= 5.8) to vaccine C (GMT-log

_{2}= 3.5). Vaccine B did not respond to this strain of BVDV-1.

_{2}= 1.0) and the second vaccine dose (GMT-log

_{2}= 6.7).

_{2}= 2.5) and C (GMT log

_{2}= 0.7) at D21. Higher response was observed in Vaccine C (GMT-log

_{2}= 6.1), followed by vaccine A (GMT-log

_{2}= 4.3), vaccine B (GMT-log

_{2}= 2.7) at D42.

## 4. Discussion

^{+}T cells, interact with DC’s membrane and complement activation [43].

## 5. Conclusions

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## Appendix

**Table A1.**Impact of vaccination against BVDV and BHV-1 with different commercial formulations in animals on a presence of cardinal signs of inflammation.

Variable | First Dose | Second Dose | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|

Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | X^{2} | Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | X^{2} | ||

Pain | 0 h ^{1} | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - |

6 h | 7 (78%) | 7 (70%) | 7 (70%) | 0 (0%) | 0.012 | 8 (89%) | 6 (60%) | 9 (90%) | 0 (0%) | 0.001 | |

24 h | 5 (56%) | 7 (70%) | 6 (60%) | 0 (0%) | 0.044 | 6 (67%) | 5 (50%) | 8 (80%) | 0 (0%) | 0.015 | |

48 h | 5 (56%) | 5 (50%) | 3 (30%) | 0 (0%) | 0.123 | 7 (78%) | 7 (70%) | 9 (90%) | 0 (0%) | 0.002 | |

72 h | 5 (56%) | 8 (80%) | 3 (30%) | 0 (0%) | 0.013 | 3 (33%) | 4 (40%) | 5 (50%) | 0 (0%) | 0.224 | |

168 h | 4 (44%) | 7 (70%) | 3 (30%) | 0 (0%) | 0.041 | 1 (11%) | 5 (50%) | 1 (10%) | 0 (0%) | 0.042 | |

Cochran’s | 0.004 | 0.000 | 0.002 | - | 0.001 | 0.001 | 0.000 | - | |||

Heat ^{6} | 0 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - |

6 h | 4 (44%) | 4 (40%) | 8 (80%) | 0 (0%) | 0.019 | 4 (44%) | 5 (50%) | 7 (70%) | 0 (0%) | 0.057 | |

24 h | 2 (22%) | 7 (70%) | 9 (90%) | 0 (0%) | 0.001 | 6 (67%) | 5 (50%) | 5 (50%) | 0 (0%) | 0.079 | |

48 h | 6 (67%) | 7 (70%) | 5 (50%) | 0 (0%) | 0.035 | 2 (22%) | 4 (40%) | 6 (60%) | 0 (0%) | 0.079 | |

72 h | 3 (33%) | 8 (80%) | 6 (60%) | 0 (0%) | 0.012 | 2 (22%) | 4 (40%) | 3 (30%) | 0 (0%) | 0.348 | |

168 h | 0 (0%) | 2 (20%) | 2 (20%) | 0 (0%) | 0.336 | 1 (11%) | 3 (30%) | 0 0%) | 0 (0%) | 0.148 | |

Cochran’s | 0.006 | 0.001 | 0.000 | - | 0.004 | 0.025 | 0.000 | - |

^{1}h: hours;

^{2}Vaccine A (n = 9);

^{3}Vaccine B (n = 10);

^{4}Vaccine C (n = 10);

^{5}Control (n = 6);

^{6}Heat: Considered 35 °C above; X

^{2}= Chi-square test. Differences were considered significant when p ≤ 0.05.

**Table A2.**Impact of systemic reaction based on respiratory score, rectal temperature, fecal consistency and sum of health score induced by vaccines against BVDV and BHV-1 with different commercial formulations.

Variables | First Dose | Second Dose | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|

Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | X^{2} | Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | X^{2} | ||

Cough | 0 h ^{1} | 0 (0%) | 0 (0%) | 1 (10%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - |

6 h | 0 (0%) | 0 (0%) | 1 (10%) | 0 (0%) | 0.616 | 1 (11%) | 2 (20%) | 1 (10%) | 0 (0%) | 0.677 | |

24 h | 0 (0%) | 1 (10%) | 2 (20%) | 0 (0%) | 0.377 | 1 (11%) | 2 (20%) | 3 (30%) | 0 (0%) | 0.441 | |

48 h | 2 (22%) | 0 (0%) | 2 (20%) | 0 (0%) | 0.133 | 1 (11%) | 4 (40%) | 1 (10%) | 1 (17%) | 0.307 | |

72 h | 0 (0%) | 3 (30%) | 1 (10%) | 1 (17%) | 0.117 | 1 (11%) | 4 (40%) | 1 (10%) | 0 (0%) | 0.199 | |

168 h | 1 (11%) | 2 (20%) | 1 (10%) | 1 (17%) | 0.915 | 1 (11%) | 2 (20%) | 3 (30%) | 0 (0%) | 0.774 | |

Cochran’s | 0.180 | 0.014 | 0.940 | 0.416 | 0.742 | 0.193 | 0.139 | 0.549 | |||

Nasal discharge | 0 h | 3 33%) | 5 (50%) | 6 (60%) | 1 (17%) | 0.331 | 4 (44%) | 5 (50%) | 6 (60%) | 0 (0%) | 0.116 |

6 h | 4 (44%) | 7 (70%) | 6 (60%) | 1 (17%) | 0.124 | 7 (78%) | 5 (50%) | 9 (90%) | 3 (50%) | 0.169 | |

24 h | 5 (55%) | 6 (60%) | 6 (60%) | 2 (33%) | 0.722 | 5 (56%) | 8 (80%) | 7 (70%) | 3 (50%) | 0.561 | |

48 h | 5 (55%) | 6 (60%) | 7 (70%) | 2 (33%) | 0.551 | 5 (56%) | 9 (90%) | 10 (100%) | 5 (83%) | 0.067 | |

72 h | 3 (33%) | 5 (50%) | 6 (60%) | 1 (17%) | 0.331 | 6 (67%) | 10 (100%) | 10 (100%) | 4 (67%) | 0.051 | |

168 h | 6 (66%) | 6 (60%) | 7 (70%) | 3 (50%) | 0.865 | 5 (56%) | 7 (70%) | 9 (90%) | 2 (33%) | 0.116 | |

Cochran’s | 0.539 | 0.920 | 0.974 | 0.709 | 0.335 | 0.009 | 0.052 | 0.044 | |||

Ocular discharge | 0 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 4 (40%) | 1 (10%) | 1 (17%) | 0.117 |

6 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 2 (22%) | 4 (40%) | 2 (20%) | 1 (17%) | 0.742 | |

24 h | 3 (33%) | 2 (20%) | 0 (0%) | 1 (17%) | 0.116 | 2 (22%) | 5 (50%) | 5 (50%) | 2 (33%) | 0.546 | |

48 h | 2 (22%) | 2 (20%) | 3 (30%) | 1 (17%) | 0.926 | 4 (44%) | 8 (80%) | 8 (80%) | 3 (50%) | 0.233 | |

72 h | 2 (22%) | 0 (0%) | 2 (20%) | 1 (17%) | 0.334 | 4 (44%) | 6 (60%) | 6 (60%) | 2 (33%) | 0.246 | |

168 h | 1 (11%) | 6 (60%) | 4 (40%) | 2 (33%) | 0.178 | 3 (33%) | 4 (40%) | 3 (30%) | 1 (17%) | 0.808 | |

Cochran’s | 0.079 | 0.002 | 0.018 | 0.543 | 0.218 | 0.155 | 0.014 | 0.649 | |||

Rectal temperature | 0 h | 4 (44%) | 1 (10%) | 7 (70%) | 0 (0%) | 0.462 | 3 (33%) | 1 (10%) | 3 (30%) | 0 (0%) | 0.152 |

6 h | 2 (22%) | 5 (50%) | 4 (40%) | 0 (0%) | 0.151 | 1 (11%) | 4 (40%) | 6 (60%) | 0 (0%) | 0.462 | |

24 h | 0 (0%) | 0 (0%) | 1 (10%) | 1 (17%) | 0.151 | 0 (0%) | 0 (0%) | 4 (40%) | 0 (0%) | 0.152 | |

48 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | |

72 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | |

168 h | 1 (11%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | |

Cochran’s | 0.569 | 0.344 | 0.278 | 0.444 | 0.101 | 0.189 | 0.152 | - | |||

Fecal consistency | 0 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - |

6 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | 1 (11%) | 0 (0%) | 1 (10%) | 0 (0%) | 0.677 | |

24 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0.462 | 0 (0%) | 1 (10%) | 0 (0%) | 0 (0%) | 0.416 | |

48 h | 3 (33%) | 0 (0%) | 0 (0%) | 2 (33%) | 0.190 | 1 (11%) | 0 (0%) | 0 (0%) | 1 (17%) | 0.661 | |

72 h | 2 (22%) | 1 (10%) | 0 (0%) | 1 (17%) | 0.211 | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | - | |

168 h | 0 (0%) | 0 (0%) | 0 (0%) | 0 (0%) | 0.396 | 2 (22%) | 0 (0%) | 0 (0%) | 0 (0%) | 0.876 | |

Cochran’s | 0.093 | 0.594 | - | 0.221 | 0.127 | 0.156 | 0.113 | 0.100 | |||

Sum of health score | 0 h | 2 (22%) | 0 (0%) | 1 (10%) | 0 (0%) | 0.300 | 1 (11%) | 3 (30%) | 1 (10%) | 0 (0%) | 0.356 |

6 h | 1 (11%) | 1 (10%) | 2 (20%) | 0 (0%) | 0.677 | 1 (11%) | 3 (30%) | 5 (50%) | 0 (0%) | 0.990 | |

24 h | 1 (11%) | 1 (10%) | 3 (30%) | 0 (0%) | 0.356 | 1 (11%) | 3 (30%) | 4 (40%) | 0 (0%) | 0.218 | |

48 h | 4 (44%) | 1 (10%) | 2 (20%) | 1 (17%) | 0.116 | 1 (11%) | 6 (60%) | 6 (60%) | 2 (33%) | 0.024 | |

72 h | 2 (22%) | 3 (30%) | 1 (10%) | 1 (17%) | 0.725 | 3 (33%) | 5 (50%) | 1 (10%) | 0 (0%) | 0.081 | |

168 h | 1 (11%) | 3 (30%) | 4 (40%) | 1 (17%) | 0.489 | 2 (22%) | 3 (30%) | 3 (30%) | 0 (0%) | 0.501 | |

Cochran’s | 0.472 | 0.119 | 0.472 | 0.629 | 0.416 | 0.501 | 0.044 | 0.044 |

^{1}h: hours;

^{2}Vaccine A (n = 9);

^{3}Vaccine B (n = 10);

^{4}Vaccine C (n = 10);

^{5}Control (n = 6); X

^{2}= Chi-square test. Differences were considered significant when p ≤ 0.05.

**Table A3.**Clinical pathology evaluated by absolute mean values (10

^{3}/µL) obtained in the counts of leukocytes, platelets and serum iron of animals vaccinated with vaccines against BVDV and BHV-1 with different commercial formulations.

Variables | First Dose | Second Dose | |||||||
---|---|---|---|---|---|---|---|---|---|

Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | ||

WBC ^{6}(10^{3}/µL) | 6 h ^{1} | 16.63 | 17.22 ^{a} | 16.07 | 13.47 | 15.89 ^{A,b,a} | 15.80 ^{A,B,a} | 18.36 ^{A,a} | 13.50 ^{B,a} |

24 h | 14.64 | 13.66 ^{b} | 16.04 | 13.60 | 13.84 ^{a} | 12.58 ^{b} | 14.40 ^{b} | 11.80 ^{a,b} | |

48 h | 14.43 | 12.19 ^{b} | 14.13 | 11.63 | 13.73 ^{a} | 12.23 ^{b} | 14.36 ^{b} | 11.45 ^{a,b} | |

72 h | 13.92 | 13.41 ^{b} | 14.30 | 12.08 | 13.51 ^{a} | 12.21 ^{b} | 14.11 ^{b} | 10.60 ^{a,b} | |

168 h | 13.70 | 13.21 ^{b} | 14.30 | 14.05 | 12.07 ^{A,B,b} | 12.20 ^{A,B,b} | 13.45 ^{A,b} | 9.62 ^{B,b} | |

LYMPH ^{7}(10^{3}/µL) | 6 h | 7.76 | 7.01 | 6.58 | 6.98 | 8.69 | 6.99 | 6.73 | 7.42 |

24 h | 8.43 | 6.91 | 6.33 | 6.13 | 8.65 | 7.01 | 5.96 | 6.65 | |

48 h | 8.25 | 6.59 | 7.39 | 6.48 | 8.40 | 6.51 | 7.08 | 6.71 | |

72 h | 8.09 | 7.37 | 7.25 | 6.82 | 8.45 | 6.96 | 7.22 | 5.98 | |

168 h | 8.11 | 7.57 | 7.67 | 7.45 | 7.78 | 6.71 | 8.19 | 5.93 | |

MONO ^{8}(10^{3}/µL) | 6 h | 2.11 ^{a} | 1.80 | 1.72 ^{a,b} | 2.06 ^{a} | 1.40 | 1.32 | 1.52 | 1.58 |

24 h | 2.06 ^{a} | 1.81 | 1.99 ^{a} | 2.22 ^{a} | 1.00 | 1.21 | 1.14 | 0.97 | |

48 h | 1.86 ^{a,b} | 1.44 | 1.71 ^{a,b} | 1.43 ^{b} | 1.31 | 1.23 | 1.25 | 1.09 | |

72 h | 1.85 ^{a,b} | 1.35 | 1.59 ^{a} | 1.23 ^{b} | 1.35 | 1.24 | 1.51 | 1.05 | |

168 h | 1.31 ^{b} | 1.38 | 1.24 ^{b} | 1.31 ^{b} | 1.02 | 1.17 | 1.33 | 0.76 | |

BAS ^{9}(10^{3}/µL) | 6 h | 0.68 ^{A,B} | 0.68 ^{A,B} | 0.41 ^{B} | 1.19 ^{A} | 0.73 | 0.90 | 0.60 | 1.11 |

24 h | 0.86 | 0.94 | 0.84 | 1.36 | 1.01 | 1.10 | 1.28 | 1.27 | |

48 h | 0.53 | 0.74 | 0.98 | 1.02 | 1.19 | 1.25 | 1.29 | 0.70 | |

72 h | 0.29 | 0.80 | 0.65 | 0.99 | 1.00 | 1.08 | 1.0 | 0.89 | |

168 h | 0.46 | 0.70 | 0.63 | 0.69 | 0.87 | 0.68 | 0.89 | 0.65 | |

NEUTR ^{10}(10^{3}/µL) | 6 h | 5.80 ^{A,B,a} | 7.38 ^{A,a} | 7.28 ^{A,a,b} | 3.26 ^{B,a,b} | 5.17 ^{B,C,a} | 6.54 ^{B,a} | 9.10 ^{A,a} | 3.43 ^{C,a,b} |

24 h | 3.21 ^{B,b} | 3.80 ^{B,b} | 6.92 ^{A,b} | 3.60 ^{B,a,b} | 2.92 ^{B,b} | 3.50 ^{B,b} | 5.83 ^{A,b,c} | 2.84 ^{B,a,b} | |

48 h | 3.70 ^{A,b} | 3.16 ^{A,b} | 3.87 ^{A,c} | 2.63 ^{A,b} | 2.91 ^{A,b} | 3.20 ^{A,b} | 4.34 ^{A,c} | 2.94 ^{A,a,b} | |

72 h | 3.54 ^{A,B,b} | 3.47 ^{A,B,b} | 4.68 ^{A,c} | 2.54 ^{B,b} | 2.63 ^{B,b} | 3.16 ^{A,B,b} | 4.17 ^{A,c} | 2.62 ^{B,b} | |

168 h | 3.68 ^{A,b} | 3.39 ^{A,b} | 4.66 ^{A,c} | 4.60 ^{A,b} | 2.49 ^{A,B,b} | 3.70 ^{A,B,b} | 3.92 ^{A,c} | 2.16 ^{B,b} | |

EOS ^{11} (10^{3}/µL) | 6 h | 0.54 ^{B,a,b,c} | 0.60 ^{B} | 0.42 ^{B,b} | 1.08 ^{A,a,b} | 0.75 ^{a,b,c} | 0.78 | 0.96 ^{a,b} | 0.96 ^{a,b} |

24 h | 0.72 ^{B,a,b,c} | 0.78 ^{B} | 0.74 ^{B,a,b} | 1.40 ^{A,a} | 1.04 ^{a} | 0.98 | 1.39 ^{a} | 1.12 ^{a,b} | |

48 h | 0.45 ^{b,c} | 0.65 | 1.10 ^{a,b} | 1.00 ^{a,b} | 1.08 ^{a} | 0.99 | 1.44 ^{a} | 0.60 ^{b} | |

72 h | 0.29 ^{B,c} | 0.73 ^{A,B} | 0.70 ^{B,a,b} | 1.40 ^{A,a} | 0.98 ^{a,b} | 0.99 | 1.06 ^{a,b} | 0.78 ^{a,b} | |

168 h | 0.40 ^{b,c} | 0.59 | 0.68 ^{a,b} | 0.57 ^{b} | 0.78 ^{a,b,c} | 0.68 | 0.81 ^{a,b} | 0.60 ^{b} | |

PLAT ^{12}(×10^{3}/µL) | 6 h | 214.33 ^{A,B} | 333.30 ^{A} | 254.20 ^{A,B} | 168.17 ^{B} | 246.56 | 221.00 | 208.80 | 188.00 |

24 h | 263.67 | 251.60 | 235.20 | 251.33 | 225.33 | 227.30 | 209.10 | 155.67 | |

48 h | 205.67 | 275.60 | 193.70 | 259.67 | 204.89 | 228.60 | 191.50 | 178.33 | |

72 h | 220.00 | 253.70 | 285.00 | 264.67 | 219.67 ^{A,B} | 241.00 ^{A} | 201.60 ^{A,B} | 157.17 ^{B} | |

168 h | 238.11 | 266.00 | 235.00 | 252.50 | 207.89 | 246.00 | 237.70 | 163.00 | |

Iron (µM/L) | 0 h | 28 ^{A} | 26 ^{A,B} | 23 ^{B} | 32 | 23 ^{A,b} | 27 ^{A,a,b} | 25 ^{A,a} | 29 ^{A,a,b} |

6 h | 27 ^{A,B} | 26 ^{B} | 21 ^{B} | 35 | 20 ^{A,B,b} | 16 ^{B,c} | 14 ^{B,b} | 25 ^{A,b} | |

24 h | 29 ^{A,B} | 23 ^{B} | 15 ^{C} | 32 | 21 ^{A,B,b} | 19 ^{B,b,c} | 12 ^{C,b} | 30 ^{A,a,b} | |

48 h | 30 ^{A,B} | 26 ^{A,B} | 23 ^{B} | 32 | 33 ^{A,b,a} | 27 ^{B,a,b} | 14 ^{C,b} | 38 ^{A,a} | |

72 h | 28 ^{A,B} | 24 ^{B,C} | 21 ^{C} | 33 | 27 ^{A,B,a,b} | 26 ^{A,B,b} | 24 ^{B,a} | 33 ^{A,a,b} |

^{1}h: hours; 1h: hours;

^{2}Vaccine A (n = 9);

^{3}Vaccine B (n = 10);

^{4}Vaccine C (n = 10);

^{5}Control (n = 6);

^{6}WBC: Total leukocytes;

^{7}LYMPH: Lymphocytes;

^{8}MONO: Monocytes;

^{9}BAS basophils;

^{10}NEUTR: Neutrophils;

^{11}EOS: Eosinophils;

^{12}PLAT: Platelets; capital letters on the same line show difference between treatments; lowercase letters in the same column show difference between times. Differences were considered significant when p ≤ 0.05.

**Table A4.**Clinical pathology evaluated by relative mean values (%) obtained in the leukogram of animals vaccinated with vaccines against BVDV and BHV-1 with different commercial formulations.

Variables | First Dose | Second Dose | |||||||
---|---|---|---|---|---|---|---|---|---|

Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | Vaccine A ^{2} | Vaccine B ^{3} | Vaccine C ^{4} | Control ^{5} | ||

LYMPH ^{6}(%) | 6 h^{1} | 46.33 ^{a} | 41.70 ^{a} | 42.00 ^{a,b} | 50.33 ^{a,b,c} | 51.78 ^{A,b,a} | 44.70 ^{B,C,a} | 36.70 ^{C,a} | 55.33 ^{A} |

24 h | 56.56 ^{A,a,b} | 49.30 ^{B,a,b} | 39.50 ^{C,a} | 44.17 ^{B,C,a} | 61.44 ^{A,a,b} | 56.10 ^{A,b,c} | 41.20 ^{B,a,b} | 57.33 ^{A} | |

48 h | 57.22 ^{a,b} | 54.20 ^{b} | 52.30 ^{c} | 56.00 ^{b,c} | 59.44 ^{a,b} | 52.90 ^{a,b,c} | 49.40 ^{b,c} | 59.00 | |

72 h | 58.56 ^{b} | 55.90 ^{b} | 50.80 ^{b,c} | 57.33 ^{b,c} | 61.11 ^{a,b} | 57.20 ^{c} | 51.70 ^{c} | 56.67 | |

168 h | 56.00 ^{a,b} | 56.40 ^{b} | 53.60 ^{c} | 55.67 ^{a,b} | 63.33 ^{b} | 55.30 ^{c} | 58.90 ^{c} | 63.17 | |

MONO ^{7}(%) | 6 h | 12.44 ^{A,B,a,b} | 10.30 ^{A,a} | 11.20 ^{A,a,b} | 15.83 ^{C,a,b} | 9.22 | 8.70 | 8.70 | 11.33 |

24 h | 13.89 ^{A,B,C,a} | 13.80 ^{A,B,C,b} | 13.00 ^{A,B,C,a} | 16.67 ^{C,b} | 7.33 | 9.90 | 7.70 | 8.00 | |

48 h | 12.44 ^{a,b} | 11.50 ^{a,b} | 11.80 ^{a,b} | 12.33 ^{a,c,d} | 9.56 | 10.30 | 8.60 | 9.67 | |

72 h | 13.22 ^{A,a,b} | 9.40 ^{B,a} | 11.00 ^{A,B,a,b} | 10.17 ^{A,B,c,d} | 9.78 | 9.90 | 10.90 | 9.67 | |

168 h | 9.67 ^{b} | 10.90 ^{a,b} | 9.40 ^{b} | 9.67 ^{d} | 8.78 | 9.80 | 9.30 | 8.00 | |

BAS ^{8}(%) | 6 h | 0.56 ^{a} | 0.50 | 0.40 | 0.67 | 0.45 ^{A,a} | 0.50 ^{A,B,a} | 0.30 ^{A,B,a} | 0.83 ^{B,}ª |

24 h | 1.11 ^{b} | 0.90 | 0.50 | 0.61 | 0.67 ^{A,a,b} | 0.70 ^{A,a} | 0.60 ^{A,a} | 1.67 ^{B,}ª | |

48 h | 0.37 ^{a} | 0.80 | 0.30 | 0.83 | 0.44 ^{A,a} | 1.80 ^{B,b} | 1.50 ^{B,b} | 1.00 ^{A,B,a} | |

72 h | 0.67 ^{a} | 0.90 | 0.70 | 0.67 | 1.33 ^{b} | 0.70 ^{a} | 1.00 ^{a,b} | 1.50ª | |

168 h | 0.67 ^{a} | 0.60 | 0.30 | 0.96 | 0.44 ^{A,a} | 1.10 ^{A,B,a,b} | 0.70 ^{A,B,a} | 1.50 ^{B,a} | |

NEUTR ^{9}(%) | 6 h | 36.80 ^{A,a} | 42.81 ^{A,a} | 43.40 ^{A,a} | 25.00 ^{B} | 35.00 ^{B,a,b} | 41.36 ^{A,B,a} | 49.50 ^{A,a} | 25.33 ^{C} |

24 h | 23.80 ^{B,c,d,e} | 30.00 ^{B,b} | 42.70 ^{A,a} | 27.00 ^{B} | 21.70 ^{B,c,d,e} | 27.81 ^{B,b} | 41.30 ^{A,a} | 23.67 ^{B} | |

48 h | 27.00 ^{a,b,c,d} | 26.00 ^{b} | 26.90 ^{b} | 22.67 | 22.40 ^{c,d,e} | 27.18 ^{b} | 30.90 ^{b} | 25.00 ^{A} | |

72 h | 25.70 ^{A,B,b,c} | 26.00 ^{A,B,b} | 31.90 ^{A,b} | 21.17 ^{B} | 20.10 ^{e} | 25.54 ^{b} | 29.40 ^{b} | 24.33 | |

168 h | 30.20 ^{a,b,c} | 26.18 ^{b} | 31.60 ^{b} | 29.16 | 21.30 ^{B,d,e} | 29.00 ^{A,b} | 25.20 ^{A,B,b} | 21.66 ^{B} | |

EOS ^{10}(%) | 6 h | 3.30 ^{B,b,c} | 3.54 ^{B,c} | 2.90 ^{B,b} | 8.78 ^{A,a,b,c} | 5.10 ^{B,a,b,c} | 4.81 ^{B,b,c} | 5.00 ^{B,a,b} | 7.16 ^{A,a,b,c} |

24 h | 5.00 ^{B,a,b,c} | 5.45 ^{B,a,b,c} | 4.70 ^{B,a,b} | 9.96 ^{A,a,b} | 8.20 ^{a,b} | 7.64 ^{a,b} | 9.10 ^{a} | 9.33 ^{a,b,c} | |

48 h | 3.30 ^{b,c} | 5.82 ^{a,b,c} | 8.20 ^{a} | 8.16 ^{a,b,c} | 8.70 ^{A,a} | 8.36 ^{A,a} | 9.40 ^{A,a} | 5.33 ^{B,A,c} | |

72 h | 2.20 ^{B,c} | 5.55 ^{B,a,b,c} | 5.50 ^{B,a,b} | 10.12 ^{A,a} | 8.30 ^{a} | 8.09 ^{a,b} | 7.10 ^{a,b} | 7.83 ^{a,b,c} | |

168 h | 3.10 ^{c} | 5.09 ^{a,b,c} | 5.50 ^{a,b} | 5.30 ^{A,c} | 7.00 ^{A,a,b,c} | 5.91 ^{B,a,b,c} | 5.80 ^{B,a,b} | 5.67 ^{B,b,c} |

^{1}h: hours;

^{2}Vaccine A (n = 9);

^{3}Vaccine B (n = 10);

^{4}Vaccine C (n = 10);

^{5}Control (n = 6);

^{6}LYMPH: Lymphocytes;

^{7}MONO: Monocytes;

^{8}BAS basophils;

^{9}NEUTR: Neutrophils;

^{10}EOS: Eosinophils; capital letters on the same line show difference between treatments; lowercase letters in the same column show difference between times. Differences were considered significant when p ≤ 0.05.

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**Figure 2.**The figure represents the mean values and standard deviations of local vaccine reaction with skin thickness at the injection site in animals vaccinated with three different formulation adjuvanted vaccines against BVDV and BHV-1. Overall statistical significance (p ≤ 0.05) is indicated in the plot as follows: Effects of treatment (P

_{tr}), day (P

_{Day}) and interactions (P

_{tr}× P

_{Day}).

**Figure 3.**The figure represents the mean values and standard deviations of systemic vaccine by rectal temperature (°C) in animals vaccinated with three different formulation adjuvanted vaccines against BVDV and BHV-1.Overall statistical significance (p ≤ 0.05) is indicated in the plot as follows: Effects of treatment (P

_{tr}), day (P

_{Day}) and interactions (P

_{tr}× P

_{Day}).

**Figure 4.**Effect of vaccination on Clinical pathology: (

**a**) Total leukocytes (×10

^{3}/µL); (

**b**) lymphocytes (%); (

**c**) monocytes (%); (

**d**) basophils (%); (

**e**) neutrophils; (

**f**) eosinophils (%), (

**g**) and platelets (×10

^{3}/µL) in animals vaccinated with three different formulation adjuvanted vaccines against BVDV and BHV-1. Reported are the mean values and standard deviations. Overall statistical significance (p ≤ 0.05) is indicated in the plot as follows: effects of treatment (P

_{tr}), day (P

_{Day}) and interactions (P

_{tr}× P

_{Day}).

**Figure 5.**Clinical pathology by serum iron levels (µM/L) in animals vaccinated with three different formulation adjuvanted vaccines against BVDV and BHV-1. Reported are the mean values and standard deviations. Overall statistical significance (p ≤ 0.05) is indicated in the plot as follows: Effects of treatment (P

_{tr}), day (P

_{Day}) and interactions (P

_{tr}x P

_{Day}).

**Figure 6.**Acute phase protein production from haptoglobin concentrations (mg/dL) in animals vaccinated with three different formulation adjuvanted vaccines against BVDV and BHV-1. Reported are the mean values and standard deviations. Overall statistical significance (p ≤ 0.05) is indicated in the plot as follows: Effects of treatment (P

_{tr}), day (P

_{Day}) and interactions (P

_{tr}× P

_{Day}).

Groups | Adjuvant Composed | Strains | Others Antigens |
---|---|---|---|

Vaccine A (n = 9) | Aluminum hydroxide (Alhydrogel^{®}) | Bovine viral diarrhea virus (BVDV)-1 (Singer) and BVDV-2 inactivated, strains from INTA ^{1} and CEVAN ^{2};bovine herpesvirus 1 (BHV)-1 (Los Angeles) inactivated | Campylobacter fetus; Campylobacter fetus veneralis; Leptospira interrogans pomona; Histophilus somni. |

Vaccine B (n = 10) | Oil-in-water adjuvant (mineral oil-based) | BVDV-1 and BVDV 2 (inactivated); BHV-1 and BoHV-5 (inactivated) | Leptospirahardjo, L. icterohaemorrhagiae, L. Bratislava, L. Pomona and L. wolfi |

Vaccine C (n = 10) | Amphigen and Quil A cholesterol and dimethyl dioctadecylammonium (DDA) bromide (QAD) adjuvant ^{®} | BVDV-1 (5960) and BVDV-2 (53,637) inactivated; BHV-1 (RBL106) thermosensitive | Leptospira canicola, L. grippotyphosa, L. hardjo, L. icterohaemorrhagiae and L. pomona |

Control (n = 6) | - | Saline Solution | - |

^{1}INTA=Instituto Nacional de Tecnología Agropecuaria, Argentina;

^{2}CEVAN = Centro de Virología Animal, Argentina, BVDV = Bovine Viral Diarrhea Virus, BHV-1 = bovine herpesvirus type 1.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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**MDPI and ACS Style**

Costa Baccili, C.; Martin, C.C.; Decaris, N.; Medici Madureira, K.; Chase, C.; Gomes, V.
Effects of 3 Different Commercial Vaccines Formulations against BVDV and BHV-1 on the Inflammatory Response of Holstein Heifers. *Vet. Sci.* **2019**, *6*, 69.
https://doi.org/10.3390/vetsci6030069

**AMA Style**

Costa Baccili C, Martin CC, Decaris N, Medici Madureira K, Chase C, Gomes V.
Effects of 3 Different Commercial Vaccines Formulations against BVDV and BHV-1 on the Inflammatory Response of Holstein Heifers. *Veterinary Sciences*. 2019; 6(3):69.
https://doi.org/10.3390/vetsci6030069

**Chicago/Turabian Style**

Costa Baccili, Camila, Camila Cecilia Martin, Nathália Decaris, Karina Medici Madureira, Christopher Chase, and Viviani Gomes.
2019. "Effects of 3 Different Commercial Vaccines Formulations against BVDV and BHV-1 on the Inflammatory Response of Holstein Heifers" *Veterinary Sciences* 6, no. 3: 69.
https://doi.org/10.3390/vetsci6030069