Foals are born immunologically naïve because transplacental transfer of antibodies in utero is extremely limited if it occurs at all. Consequently, for immune protection, a newborn foal is entirely dependent on maternal antibodies that are absorbed following ingestion of colostrum from the dam during the first few hours of life. Failure of this passive transfer of maternal antibodies puts a foal at considerable risk for development of infectious diseases. The degree of risk depends on multiple factors, including management practices and pathogen exposure potential. Plasma concentrations of immunoglobulins (specifically IgG) in foals that develop septicemia are significantly lower than concentrations in unaffected foals.1 Complete FPT in foals has been defined as plasma IgG concentrations of < 200 mg/dL2; PFPT has been described as plasma IgG concentrations of 200 to 400 mg/dL or 200 to 800 mg/dL.2,3 Plasma IgG concentrations ≥ 400 mg/dL may be adequate for healthy foals in a clean environment with minimal exposure to pathogens, but concentrations ≥ 800 mg/dL are considered optimal.3,4 A plasma concentration of IgG between 400 and 800 mg/dL may be inadequate for a compromised or systemically ill foal.1,5
Among horses and ponies, the incidence of FPT and PFPT ranges from 10% to 20% and 17% to 19%, respectively.6–10 Incidence rates of FPT and PFPT were reported to be higher (30% and 27%, respectively) in a population of hospitalized foals.5 The most important risk factor for development of FPT in foals appears to be poor-quality colostrum (ie, IgG concentration < 1,000 mg/dL).8,10–12 Other mare- or foal-associated factors associated with FPT include agalactia, premature lactation, inability or lack of desire to nurse, prematurity, dysmaturity, foal rejection by the mare, and failure to absorb colostral immunoglobulins.3,4
Monitoring the success of passive transfer by measuring concentrations of immunoglobulins in the blood of neonatal foals is critical if early intervention and treatment are required. Various screening tests have been developed over the years for evaluation of plasma IgG concentrations in foals.6,13–15 An optimal screening test for passive transfer success in neonatal foals would be rapid, accurate, repeatable, quantitative, and cost-effective. The objective of the study of this report was to validate a TIA for measurement of plasma IgG concentrations in foals.
Materials and Methods
Thirty-six foals born at the Equine Reproduction Laboratory, Colorado State University, were included in the study. Blood samples (2.0 to 5.0 mL) were collected from foals at 12 and 24 to 36 hours after birth as part of routine postnatal care. In addition, samples were collected from 10 of the foals prior to nursing. Unless otherwise stated, blood samples were collected by use of a 5.0-mL syringe and 20-gauge needle and immediately transferred into an evacuated tube containing the anticoagulant EDTA. The samples were centrifuged and the plasma transferred into a sterile cryogenic viala and either refrigerated or frozen (−20°C) until analyzed for IgG content. All foals were privately owned, and samples were collected with consent of owners.
Assessment of the effect of serial dilution of plasma on measurement of IgG concentration—Samples of plasma obtained from 6 foals at either 12 or 24 hours after birth underwent serial dilution with plasma obtained from foals before suckling (presuckle plasma). The initial plasma sample (200 μL) containing high antibody concentration was diluted 1:1 with presuckle plasma (200 μL) containing < 100 mg of IgG/dL as measured by use of the RID assay. The resultant diluted sample was subsequently diluted 1:1 again with presuckle plasma. The dilution procedure was repeated until five 1:1 dilutions had been performed and diluted samples collected. The IgG contents of the undiluted and all diluted samples (n = 54) were measured via single RID assay and the TIA.
Comparison of IgG concentrations in plasma and serum samples by use of the TIA—Blood samples were collected in a 10-mL syringe from each of 6 foals at 1 to 3 time periods each (before suckling, 12 hours after birth, and 24 to 36 hours after birth). Five milliliters of blood was immediately transferred into each of 2 evacuated tubes: 1 without anticoagulant and 1 containing EDTA. The sample in the tube without anticoagulant was allowed to clot at room temperature (approx 22°C). Each aliquot was subsequently centrifuged, and the serum or plasma was transferred into a labeled cryogenic vial and frozen (–20°C) until analyzed for IgG content via the TIA.
Assessment of the repeatability of plasma IgG concentration measurement—Plasma samples from 2 foals with either medium (determined 12 hours after birth) or high (determined 24 to 36 hours after birth) IgG concentrations (based on RID assay results) were evaluated via the TIA 6 times in succession on the same day. An additional series of tests was performed on plasma samples from an additional 2 foals with either medium (determined 16 hours after birth) or high (determined 36 hours after birth) IgG concentrations. Immunoglobulin G concentrations were measured in each sample via the TIA once per day on 5 days. Repeatability tests were performed to evaluate coefficient of variation of the assay.
Assessment of the effect of ambient temperature on assay results—Three plasma samples of known IgG concentrations were assayed in duplicate via the TIA at 3 temperatures (8°C, 22°C, and 37°C) to determine the effect of ambient temperature on assay results.
Evaluation of plasma IgG concentration in foals at 3 time periods after birth—Blood samples were collected from 36 foals at various intervals after birth. Samples from 10 foals were collected prior to nursing, at 12 to 18 hours after birth, and at 24 to 36 hours after birth. Samples from 26 additional foals were only collected once between 12 and 36 hours after birth. All plasma samples were evaluated for IgG concentration via RID assay and TIA.
All RID assays were performed at the Colorado Veterinary Diagnostic Laboratory, Fort Collins, Colo, by use of a commercially available kit.b The RID assay was performed according to the manufacturer's instructions; 3 μL of serum or plasma was assessed in each test. Sample precipitation rings were evaluated after approximately 18 hours of incubation at room temperature.
All materials used in the TIAc were allowed to equilibrate to room temperature before proceeding with an evaluation. For each assay, 25 μL of foal plasma was added to 1.5 mL of dilution buffer in a 4-mL cuvette. Plasma and buffer were gently mixed by inverting the cuvette 8 to 10 times. Next, 180 μL of the diluted plasma was added to 1.3 mL of a polymer buffer contained in a second cuvette. This combination was subsequently mixed by inverting the cuvette 8 to 10 times. The second cuvette was inserted into a spectrophotometer,d and the system was zeroed. The cuvette was then removed from the spectrophotometer, and 180 μL of goat anti-equine IgG antiserume was added. The cuvette was sealed and gently inverted 8 to 10 times to mix the contents. The cuvette was then replaced into the spectrophotometer, and the value of %T was evaluated.
A calibration curve was generated by comparing plasma IgG concentrations determined via the RID assay against %T values obtained via the TIA. The spectrophotometer was subsequently programmed to convert %T values to milligrams per deciliter and display the calculated IgG concentration (mg/dL).
Data analysis—With regard to determination of plasma IgG concentrations, sensitivity, specificity, and positive and negative predictive values for the TIA were calculated with RID assay–derived IgG concentrations as the reference standard. Data for all the undiluted and diluted plasma samples that were used for the assessment of the effect of serial dilution of plasma on measurement of IgG concentration by both assays were analyzed. Sensitivity was defined as the probability that a positive experimental test result was in agreement with the corresponding IgG reference test result (ie, probability that a TIA value ≤ 400 mg/dL or ≤ 800 mg/dL was in agreement with an RID assay value ≤ 400 mg/dL or ≤ 800 mg/dL, respectively). Specificity was defined as the probability that a negative experimental test result was in agreement with the corresponding IgG reference test result (ie, probability that a TIA value > 400 mg/dL or > 800 mg/dL was in agreement with an RID assay value > 400 mg/dL or > 800 mg/dL, respectively). Prevalence was the percentage of foals with FPT (plasma IgG concentration < 400 mg/dL) in the test population. Positive predictive value is the probability of disease in an animal for which test results for that disease are positive (based on the prevalence of FPT in the test population). Negative predictive value is the probability of no such disease in an animal for which test results for that disease are negative (again, based on the prevalence of FPT in the test population). Positive and negative predictive values were calculated by use of the formula reported by McClure et al.13 Linear regression analysis and coefficient of variation analysis were performed by use of a commercial statistical software package.f
Results
Effect of serial dilution of plasma on measurement of IgG concentration—Serial dilution of plasma collected from foals at 12 to 24 hours after birth with presuckle plasma resulted in decreases in %T that were highly inversely correlated (r = −0.95) with RID assay–determined plasma IgG concentrations (Figure 1). Plasma IgG concentrations determined via RID assay of 400 and 800 mg/dL were associated with TIA %T values of approximately 80.5% and 69.9%, respectively.
Comparison of IgG concentrations determined via an RID assay and a TIA in plasma samples collected from 6 foals at either 12 or 24 hours after birth (subsequently serially diluted with plasma obtained before suckling from 10 foals to provide 36 samples [diluted and undiluted]).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Comparison of IgG concentrations determined via an RID assay and a TIA in plasma samples collected from 6 foals at either 12 or 24 hours after birth (subsequently serially diluted with plasma obtained before suckling from 10 foals to provide 36 samples [diluted and undiluted]).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Comparison of IgG concentrations determined via an RID assay and a TIA in plasma samples collected from 6 foals at either 12 or 24 hours after birth (subsequently serially diluted with plasma obtained before suckling from 10 foals to provide 36 samples [diluted and undiluted]).
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Comparison of IgG concentrations in plasma and serum samples by use of the TIA—Light transmission data (%T values) obtained from analysis of serum and plasma samples were almost identical. The correlation coefficient was 0.99 (Figure 2).
Relationship between IgG concentrations in plasma and serum obtained from 6 foals at 1 to 3 time periods each (before suckling, 12 hours after birth, and 24 to 36 hours after birth) as measured by a TIA.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Relationship between IgG concentrations in plasma and serum obtained from 6 foals at 1 to 3 time periods each (before suckling, 12 hours after birth, and 24 to 36 hours after birth) as measured by a TIA.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Relationship between IgG concentrations in plasma and serum obtained from 6 foals at 1 to 3 time periods each (before suckling, 12 hours after birth, and 24 to 36 hours after birth) as measured by a TIA.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Assessment of the repeatability of plasma IgG concentration measurement—Values of %T for plasma samples that were evaluated repeatedly on the same day or on sequential days were assessed (Table 1). The coefficients of variation for the repeated tests ranged from 2.17% to 3.81%.
Concentrations of IgG in plasma obtained from foals (2/experiment) determined once by use of an RID assay and 6 times during 1 day or once during each of 5 sequential days by use of a TIA.
| Timing of multiple assessments | Foal | Time after birth (h) | RID assay IgG concentration (mg/dL) | TIA assessment (%T value) | Mean ± SD %T | CV (%) | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 3 | 2 | 4 | 5 | 6 | ||||||
| Same day | 1 | 12 | 652 | 74.8 | 71.8 | 73.5 | 73.1 | 70.4 | 75.0 | 73.0 ± 1.8 | 2.57 |
| 2 | 12 | 1,243 | 38.1 | 41.2 | 38.9 | 38.6 | 41.1 | 40.7 | 39.8 ± 1.4 | 3.52 | |
| Sequential days | 1 | > 24 | 1,439 | 70.1 | 68.8 | 70.9 | 67.0 | 69.7 | ND | 69.3 ± 1.5 | 2.17 |
| 3 | 12 | 3,798 | 39.4 | 38.9 | 38.9 | 38.0 | 42.0 | ND | 39.4 ± 1.5 | 3.81 | |
CV = Coefficient of variation. ND = Not done.
Effect of ambient temperature on TIA results—To determine the effect of ambient temperature on TIA results, 3 plasma samples of known IgG concentrations were assayed at 3 temperatures (8°C, 22°C, and 37°C). Ambient temperature did not affect %T values. At 8°C, 22°C, and 37°C, the %T value was 43.7, 46.8, and 48.1, respectively, for sample 1; 61.4, 62.8, and 60.3, respectively, for sample 2; and 73.6, 77.4, and 77.7, respectively, for sample 3.
Evaluation of plasma IgG concentration in foals at 3 time periods after birth—Plasma samples obtained from 36 foals at various intervals after birth (before nursing through 36 hours) were assessed, and results of the RID assay (mg/dL) and TIA (%T) were inversely correlated (r = −0.81). Concentrations of IgG in all plasma samples obtained prior to nursing were < 150 mg/dL (as determined via the RID assay), and those samples had a corresponding %T value > 94% (as determined via the TIA). Samples obtained 12 to 18 hours after birth had RID assay–determined IgG concentrations that ranged from 652 to 4,390 mg/dL; results of the TIA ranged from 39.9% to 75.8%. All plasma samples obtained > 24 hours after birth had IgG concentrations > 1,200 mg/dL (as determined via the RID assay) and %T values < 75% (as determined via the TIA). Comparative RID assay and TIA data from all samples analyzed in the study were used to generate a calibration curve to enable conversion of %T values to milligrams of IgG per milliliter of plasma. Plasma IgG concentrations determined via RID assay and converted TIA data (both reported as mg of IgG/mL of plasma) were compared. The relationship between RID assay data and TIA data for plasma samples with RID assay IgG concentrations < 2,000 mg/dL was assessed (Figure 3).
Comparison of IgG concentrations determined via RID assay and TIA in 36 samples of plasma obtained from foals at various intervals after birth. Samples were collected prior to nursing, at 12 to 18 hours after birth, or at 24 to 36 hours after birth. Values of %T determined via the TIA were converted to milligrams of IgG per deciliter of plasma by use of a calibration curve generated from TIA and RID assay data.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Comparison of IgG concentrations determined via RID assay and TIA in 36 samples of plasma obtained from foals at various intervals after birth. Samples were collected prior to nursing, at 12 to 18 hours after birth, or at 24 to 36 hours after birth. Values of %T determined via the TIA were converted to milligrams of IgG per deciliter of plasma by use of a calibration curve generated from TIA and RID assay data.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Comparison of IgG concentrations determined via RID assay and TIA in 36 samples of plasma obtained from foals at various intervals after birth. Samples were collected prior to nursing, at 12 to 18 hours after birth, or at 24 to 36 hours after birth. Values of %T determined via the TIA were converted to milligrams of IgG per deciliter of plasma by use of a calibration curve generated from TIA and RID assay data.
Citation: American Journal of Veterinary Research 68, 9; 10.2460/ajvr.68.9.1005
Overall evaluation of the TIA—With regard to determination of plasma IgG concentrations, sensitivity, specificity, and positive and negative predictive values for the TIA were calculated by use of the RID assay– derived IgG concentrations as the reference standard (Table 2). Data from 55 foal plasma samples obtained during the study were analyzed. The TIA sensitivity was > 90% when evaluated at both 400 and 800 mg of IgG/dL of plasma. The TIA specificity was 99.1% when evaluated at 400 mg of IgG/dL of plasma, but this decreased to 70.5% when evaluated at 800 mg of IgG/dL of plasma.
Sensitivity, specificity, and predictive values of the TIA (compared with RID assay data as the reference standard ) for assessment of IgG concentration in 55 foal plasma samples containing IgG concentrations ≤ 400 and ≤ 800 mg/dL.
| Variable | Plasma IgG concentration | |
|---|---|---|
| ≤ 400 mg/dL | ≤ 800 mg/dL | |
| Sensitivity (%) | 0.923 | 0.915 |
| Specificity (%) | 0.991 | 0.705 |
| Positive predictive value | 0.981 | 0.715 |
| Negative predictive value | 0.964 | 0.911 |
Discussion
Transplacental transfer of immunoglobulins from dam to fetus does not occur in horses because of the epitheliochorial type of placentation. As a consequence, foals are born without clinically important circulating concentrations of IgG. Antibodies from a mare's blood are selectively concentrated in colostrum during the last 2 weeks of pregnancy.4 After ingestion of colostrum, specialized enterocytes in the small intestine of a foal nonselectively absorb the colostral immunoglobulins via pinocytosis.3 Intestinal absorption of antibodies through the enterocytes is greatest during the first 6 to 8 hours after birth and essentially ceases by 24 to 36 hours as the specialized enterocytes are replaced by epithelial cells that are incapable of pinocytosis.
Presently, collection of a blood sample from a newborn foal at 8 to 12 hours after birth is recommended for evaluation of circulating IgG concentrations prior to the cessation of antibody absorption from the small intestine across the gastrointestinal tract wall.2 If plasma IgG concentration is determined to be ≥ 800 mg/dL during this early postpartum period, passive transfer is considered to be adequate in that foal, and no further testing or intervention is required. If plasma IgG concentration is low (< 400 mg/dL), a foal may be orally administered supplemental frozen-thawed colostrum or a commercial colostrum substitute with the realization that some transfer of maternal antibodies subsequent to colostrum ingestion may occur between 12 and 24 hours after birth. Alternatively, if evaluation of passive transfer status is not performed until 24 to 36 hours after birth and plasma IgG concentration is determined to be inadequate, oral supplementation will no longer be effective. Intravenous administration of equine plasma would be needed to increase IgG concentration if FPT was diagnosed in a foal > 24 hours old.
Diagnostic tests for evaluation of plasma IgG concentrations in foals include single RID assay,15–17 glutaraldehyde coagulation test,16,18 zinc sulfate turbidity test,1,15,19,20 latex agglutination test,12,16,21 enzyme immunoassay,5,13,14 and TIA.19,21,22 In addition, serum total protein concentration has been evaluated as a potential indicator of FPT.5,17 Several recent studies5,13,17 have been undertaken to compare the use of various tests for diagnosis of FPT in foals.
The RID assay is considered to be the most accurate test for quantitative measurement of serum or plasma IgG concentration in foals3,4 and is used as the reference standard when other tests are evaluated. Unfortunately, results of the RID assay are generally not available for at least 24 hours; therefore, the assay is impractical for routine use on a breeding farm or at a veterinary clinic, where rapid results are required to direct therapeutic intervention. In addition, a recent report17 indicated that there can be wide limits of agreement among commercial RID assays, especially at plasma IgG concentrations > 400 mg/ dL. Results of physical examination, CBC, and serum biochemical analyses may also be used to determine the need for medical intervention if circulating IgG concentrations cannot be immediately assessed.
Turbidimetric immunoassays are based on a specific antigen-antibody reaction in the presence of excess antibody concentration, enabling accurate quantitation of circulating IgG concentrations.19 Turbidimetric immunoassay technology (involving spectrophotometric endpoints) has been previously used for evaluation of IgG concentrations in bovine and equine serum.19,22 It has been reported22 that hemolysis does not interfere with TIAs during measurement of serum IgG concentrations.
In the present study, the advantages of the TIA evaluated were that it was easy to perform, it could be used with samples of either serum or plasma, the results were available within approximately 10 minutes, and the spectrophotometric equipment required for the assay is already present on many breeding farms and at veterinary clinics. Assay results had a high degree of repeatability when samples were tested multiple times in 1 day and when samples were tested once per day during a period of several days. Another important advantage was that data regarding circulating IgG concentrations were quantitative (ie, reported as mg/dL). Therefore, changes in plasma IgG concentrations could be determined over time as a means of monitoring foals with FPT or evaluating their responses to treatments administered PO or IV.
The TIA was accurate, relative to the standard RID assay, for detection of FPT at plasma IgG concentrations < 400 mg/dL. To some extent, the TIA underestimated IgG concentrations in some plasma samples that had IgG concentrations > 400 mg/dL (as determined via the RID assay). Consequently, PFPT (ie, 400 to 800 mg of IgG/dL of plasma) may have been diagnosed by use of the TIA in some foals for which the RID assay results indicated that plasma IgG concentrations were > 800 mg/dL. In contrast, the TIA rarely failed to accurately identify a foal with plasma IgG concentration < 800 mg/dL (as determined by the RID assay). Clabough et al16 proposed that the negative predictive value of any test for FPT in foals may be the most important variable if the primary goal is to identify foals with low circulating IgG concentrations.
Results of the present study indicated that values generated by the evaluated TIA were highly correlated with data obtained via the RID assay and that TIA-derived findings were repeatable. The TIA appeared to be most accurate in identifying foals with FPT (< 400 mg of IgG/dL of plasma). Importantly, the rapid availability of accurate quantitative results could make this test valuable in clinical situations involving FPT-affected foals, for which intervention decisions are time dependent.
ABBREVIATIONS
| FPT | Failure of passive transfer |
| PFPT | Partial failure of passive transfer |
| TIA | Turbidimetric immunoassay |
| RID | Radial immunodiffusion |
| %T | Percentage light transmission |
Nalge Co, Rochester, NY.
Immunocheck, VMRD Inc, Pullman, Wash.
ARS foal IgG test, Animal Reproduction Systems, Chino, Calif.
Densimeter model 590a, Animal Reproduction Systems, Chino, Calif.
Midland BioProducts Corp, Boone, Iowa.
SAS, version 8.0, SAS Institute Inc, Cary, NC.
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