Research Summary - 2
Measuring pathogen specificity of IgG in milk replacers and the potential for supplementing with pathogen-specific IgY
| Date/Time: |
9/12/2025
14:15
|
| Author: |
Roger L Saltman |
| Clinic: |
RLS Management Solutions LLC |
| City, State, ZIP: |
Cazenovia, NY 13035 |
R. L. Saltman, DVM, MBA
;
S. W. Jones, PhD
;
M. Tollefson, BS
;
1RLS Management Solutions LLC, Cazenovia, NY 13035
2Arkion Life Sciences, New Castle, DE 19720
Introduction:
Milk Replacers (MRs) are formulated to provide calves with the nutrients necessary for growth. Milk-based protein sources contain varying amounts of IgG that can provide the calf with enteric immunity by binding to pathogens for removal from the GI tract. There is very little literature that refers to this potential passive immunity offered by milk-based MR components and no previous work to demonstrate the relative levels of this passive immunity in specific MR formulations.
The Objective of this study was to develop a method to determine the total amount of IgG present in five commercial MRs as well as the specificity to common causes of calf scours. Next, the improvement in specific antibody titers was evaluated by adding IgY, the avian equivalent to IgG, to the MR. The IgY egg powder (EggTek®-C) was harvested from the eggs of chickens inoculated with multiple killed enteric calf pathogens.
Materials and methods:
Five commercial MRs were randomized and reconstituted following manufacturers’ instructions. Total IgG titers were measured using a Bovine IgG ELISA Kit (ICL, Inc.). IgG was purified from each MR by: 1) acidifying the sample to remove casein, 2) precipitating the IgG with ammonium sulfate, 3) purification through a Protein A column, and 4) dialysis against PBS. The purified IgG was then conjugated to a horseradish peroxidase (HRP, Abcam Ltd) and used in direct ELISAs to measure antigen specificity against: bovine rotavirus, bovine coronavirus, Crypto. parvum, E. coli, and Salmonella Dublin. The amount of IgG tested for each MR was equivalent to a 10 oz. dose of MR. Specificity results were determined using A450 values (absorbance at 450 nm) providing a measure of the amount of antibody bound to the pathogen. ANOVA comparison of least significant difference was performed at a significance level of 0.05. IgY was then purified from the egg powder and labeled with HRP. The equivalent of a 2 g dose of the IgY egg powder was added to each MR and assayed against the same pathogens. Paired T-Tests were performed at a significance level of 0.05.
Results:
Total IgG (mg/g MR) varied among the MRs with values of 2.01 mg/g in MR B, 3.76 mg/g in MR C, 5.65 mg/g in MR D, 11.09 mg/g in MR A, and 12.16 mg/g in MR E. All levels were significantly different except MR A was not different from MR E. The antigen specific A450 values also varied significantly among the MRs, but surprisingly, they did not necessarily correlate with the total IgG levels. For example, the A450 value against bovine rotavirus were 0.009 for MR B, 0.031 for MR C, 0.128 for MR D, 0.136 for MR A, and 0.301 for MR E. Though the total IgY of MR A and MR E were relatively close, the specific bovine rotavirus titer in MR A was more comparable to MR D. This was seen for all the antigens, where MR D generally had numerically higher A450 values than MR A.
When the equivalent of 2 g of the IgY egg powder was added to the 10 oz dose of MRs, the specific A450 values improved for all antigens and MRs. The increase in A450 was statistically significant (p-value < 0.05) for all antigens and MRs except for Salmonella Dublin for MR E, which increased from 0.437 to 0.478 with IgY. For the other antigens, the magnitude of the A450 increase depended on the MR. For example, total Ig against E. coli for MR A increased from 0.131 to 0.401 (a 200% increase), while E. coli Ig for MR E increased from 0.494 to 0.643 (a 30% increase). MR B had the highest average improvements (~2,000%), followed by MR C (~600% improvement), MR A (~150% improvement), MR D (~90% improvement), and MR E (~20% improvement).
Significance:
This analysis highlights the importance of testing not only the total IgG level but the specificity of the IgG to ensure potential effectiveness against enteric pathogens. Results show that not all protein sources or MRs are the same with some having more IgG against enteric pathogens than others. The addition of a hyperimmunized egg powder to MRs can significantly improve specific titers for enteric pathogens.
