Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves
Abstract
:1. Introduction
2. Materials and Methods
2.1. Egg Powder Enriched with IgY Abs to BCoV
2.2. BCoV IgY Specific Antibody ELISA
2.3. Fluorescent Focus Reduction virus Neutralization Test
2.4. Virus and Inoculum Production
2.5. Experimental Design to Test the Efficacy of Egg Yolk Powder to Prevent BCoV Diarrhea in Calves
2.6. Coronavirus Antigen Detection
2.7. Quantitative Real-Time BCoV PCR
2.8. Cell Culture Immunofluorescence Assay (CCIF)
2.9. Bovine Isotype-Specific Antibody ELISA
2.10. Statistical Analysis
3. Results
3.1. Development of a Product Based on IgY Abs to BCoV
3.2. Quality Control of Egg Yolk Powder DS Batches Enriched in IgY to BCoV
3.3. BCoV IgY-Specific Antibody ELISA Validation
3.3.1. Positive Control: Admission Range and Linearity
3.3.2. ROC Curve: Cut-Off Value and Associated Sensitivity and Specificity
3.3.3. Assay Intermediate Precision
3.3.4. Correlation between the IgY ELISA Ab Titers and Neutralizing Ab Titers
3.4. Reproduction of BCoV Infection and Diarrhea in Colostrum-Deprived Calves
3.5. Egg Powder Containing BCoV IgY Abs Reduced Diarrhea Duration and Severity in a Colostrum-Deprived Calf Model
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Product | DS Batch | ELISA | VN (Mebus) | VN (Arg95) | Humidity 1 (%) | Fat 1(%) | Proteins 1 (%) | Coliforms 2 (CFU/g) | Salmonella 2 (P-A/25 g) |
---|---|---|---|---|---|---|---|---|---|
IgY to BCoV | 1 | 1024 | 64 | 64 | 2.97 | 39.63 | 48.31 | <10 | A |
IgY to BCoV | 2 | 1024 | 64 | 64 | 3.85 | 40.61 | 47.06 | <10 | A |
supplemented Milk | 512 | 32 | 32 | - | - | - | - | - |
Parameter | Result | Acceptance Criteria | |
---|---|---|---|
Accuracy and precision measurements | IgY in avian and bovine samples | ||
Associated parameters | ODc 405 nm | P% | |
Threshold (cut-off) | >0.110 | 18.68% | ODc = 0.100 P% 13–20% |
Diagnosis Specificity (DSp) | 98.2% | >90% | |
Diagnosis Sensitivity (DSe) | 97.7% | >90% | |
Intermediate precision Coefficient of Variation (CV) | 27% (Figure 3B) | <30% | |
Linearity | YES R2 = 0.91 p = 0.0001 | Linear relationship between OD and sample dilution | |
Reference standard reagent | Sera from chicken immunized with experimental vaccines with known BCoV antigen concentration. | ||
Operating range of assay | Starting sample dilution 1:32 Linearity was demonstrated between 1024–65536 reciprocal dilutions (Figure 3C) | ||
“Specificity” | The assay detects IgY antibodies against BCoV but not to Rotavirus, Salmonella or Escherichia coli | No cross reaction against NCD vaccine antigens |
IgY ELISA (Cut-Off: 18.68 P%) Sample Dilution 1/256 | |||
---|---|---|---|
Reference Population | Negative 0 | Positive 1 | |
Negative (0) | 108 | 5 | 113 (46.9%) |
Positive (1) | 3 | 125 | 128 (53.1%) |
111 (46.1%) | 130 (53.9%) | 241 | |
Kappa | 0.933 | ||
Standard error | 0.023 | ||
95% CI | 0.888 to 0.979 |
Sample | BCoV Infectious Titer (FFU/mL) |
---|---|
Inoculum | 105 |
SIC | 106 |
Colon | 109 |
LIC | 1010 |
Feces | 1010 |
Diarrhea | Shedding of Infectious Virus | |||||||
---|---|---|---|---|---|---|---|---|
Treatment Group | n | Onset (PID) | Duration (Days) | Severity (AUC) | Hyperthermia (Days) | Onset (PID) | Duration (Days) | Magnitude (AUC) |
Gp 1: BCoV-IgY | 4 | 6.25 A | 3.8 B | 17.7 B | 1.5 B | 6.3 A | 3.0 B | 23.8 A |
Gp 2: Control | 4 | 1.5 B | 7.8 A | 25.4 A | 4.75 A | 2.0 B | 6.0 A | 36.4 A |
p-value | 0.0019 | 0.0003 | 0.0036 | - | 0.0041 | 0.0104 | 0.103 |
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Bok, M.; Vega, C.G.; Castells, M.; Colina, R.; Wigdorovitz, A.; Parreño, V. Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves. Viruses 2023, 15, 708. https://doi.org/10.3390/v15030708
Bok M, Vega CG, Castells M, Colina R, Wigdorovitz A, Parreño V. Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves. Viruses. 2023; 15(3):708. https://doi.org/10.3390/v15030708
Chicago/Turabian StyleBok, Marina, Celina G. Vega, Matias Castells, Rodney Colina, Andrés Wigdorovitz, and Viviana Parreño. 2023. "Development of an IgY-Based Treatment to Control Bovine Coronavirus Diarrhea in Dairy Calves" Viruses 15, no. 3: 708. https://doi.org/10.3390/v15030708