Determination of colostrum quality using Brix refractometer in sheep.

In this study, we aimed to determine the quality of colostrum in sheep by using Brix refractometer. The research included 100 sheep of Merino X Kivircik crossbred. From each, we collected 15 mL of colostrum samples in falcon tubes within the first 8 h after delivery. Mean colostral IgG level of sheep was 156.68 ± 7.23 g L-1, optical and digital Brix refractometer values (%) were determined as 27.43 ± 0.53 and 27.69 ± 0.60, respectively. Ewes carrying twin lambs produced significantly higher quality colostrum than those carrying single lambs. However, parity did not affect the colostrum quality. Optical and digital Brix values were correlated with gold standard radial immunodiffusion (RID) colostral IgG level (r = 0.70 and r = 0.64, respectively). Also, optical and digital Brix refractometers were found to be highly correlated (r = 0.98, P < 0.001). While the optimal Brix value was 22% for the 50, 60 and 70 g L-1 IgG threshold values (by means of RID as the potential good quality threshold value for ewe colostrum quality), this value was 23% for 80 g L-1. We can conclude that Brix refractometers is a valuable tool for determining ewe colostrum quality. A cut point of 22% Brix for defining good quality colostrum in ewes was most appropriate for our data.

Invited review: The importance of colostrum in the newborn dairy calf.

It is critical that bovine maternal colostrum is fed to newborn calves during their first hours of life. Colostrum is the secretion a cow produces after mammary involution that is rich in various nutrients. In addition to the nutritive value for newborn calves, immunoglobulins are of interest due to their role in developing the naïve immune system of calves at birth. The process by which a calf acquires immunity via absorption of immunoglobulins is defined as passive immunity. When calves consume an adequate amount of immunoglobulins, they are classified as having successful passive immunity (SPI). In contrast, if they are deprived of adequate colostrum, they are considered to have had a failure of transfer of passive immunity (FPI). Transfer of passive immunity is assessed by measuring serum IgG concentrations at 24 to 48 h of age. The major factors that influence whether a calf has SPI or FPI are colostrum IgG concentration, quantity fed, and age of calf at colostrum feeding. Monitoring apparent efficiency of immunoglobulin absorption in calves is often recommended to evaluate overall colostrum management practices. Serum IgG analyses can be determined with direct (radial immunodiffusion) or indirect (refractometry) methods and used to assess SPI or FPI prevalence.

Determination of immunoglobulin concentrations and genetic parameters for colostrum and calf serum in Charolais animals.

Colostrum samples from 366 Charolais primiparous cows, as well as serum from their calves at 24 to 48 h of age, were collected to gain an overview of the situation regarding passive immune transfer in beef cattle, from both the phenotypic and genetic points of view. All samples were analyzed to quantify their G1 immunoglobulins by radial immunodiffusion (RID) and their IgG, IgA, and IgM using ELISA. The average concentrations obtained in colostrum were 84 mg/mL for RID-IgG1, and 158 mg/mL, 4.5 mg/mL and 10.8 mg/mL for ELISA-IgG, -IgA, and -IgM, respectively. The corresponding values in calf serum were 19.9, 30.6, 1.0, and 1.9 mg/mL. Apart from the general environmental effect (farm-year combination and laboratory conditions), the characteristics of the dams tested did not reveal any influence on colostrum immunoglobulin concentrations. Calving difficulty, as well as the birth weight and sex of calves, were found to be associated with serum concentrations in some cases. Heritability estimates were low to moderate, with the highest being for RID-IgG1 in colostrum (h2 = 0.28, standard error = 0.14) and serum (h2 = 0.36, standard error = 0.18). Phenotypic correlations among the different immunoglobulins were generally positive or null, and none of the genetic correlations were significant due to large standard errors. The phenotypic correlation between dam colostrum and calf serum values was 0.2 for RID-IgG1 and null for the 3 ELISA measurements. The correlation between RID-IgG1 and ELISA-IgG was, unexpectedly, null for colostrum and 0.4 for serum. Increased RID-IgG1 levels in calf serum were associated with improved survival, as well as better early growth and fewer health problems. These results thus showed that despite generally higher concentrations in beef than in dairy cattle, passive transfer was unsuccessful in a considerable number of calves. This should be brought to the attention of breeders to avoid negative effects on survival and subsequent performance. The heritability estimates were encouraging; however, obtaining phenotypes on a large scale constitutes a real limitation regarding these traits.

Accuracy of direct and indirect methods for assessing bovine colostrum quality using a latent class model fit within a Bayesian framework.

Feeding high-quality colostrum is essential for calf health and future productivity. Therefore, accurate assessment of colostrum quality is a key component of dairy farm management plans. Direct and indirect methods are available for assessment of colostrum quality; however, the indirect methods are rapid, inexpensive, and can be performed under field settings. A hierarchical latent class model fit within a Bayesian framework was used to estimate the sensitivity (Se) and specificity (Sp) of the radial immunodiffusion (RID) assay, transmission infrared (TIR) spectroscopy, and digital Brix refractometer for the assessment of low-quality bovine colostrum in Atlantic Canada dairy herds. The secondary objective of the study was to describe the distribution of herd prevalence of low-quality colostrum. Colostrum quality of 591 samples from 42 commercial Holstein dairy herds in 4 Atlantic Canada provinces was assessed using RID, TIR spectroscopy, and digital Brix refractometer. The accuracy of all tests at different Brix value thresholds was estimated using Bayesian latent class models to obtain posterior estimates [medians and 95% Bayesian credibility intervals (95% BCI)] for each parameter. Using a threshold of <23% for digital Brix refractometer and <50 g/L for RID and TIR spectroscopy, median (95% BCI) Se estimates were 73.2 (68.4-77.7), 86.2 (80.6-91.0), and 91.9% (89.0-94.2), respectively. Median (95% BCI) Sp estimates were 85.2% (81.0-88.9) for digital Brix refractometer, 99.4% (97.0-100) for RID, and 90.7% (87.8-93.2) for TIR spectroscopy. Median (95% BCI) within-herd low-quality colostrum prevalence was estimated at 32.5% (27.9-37.4). In conclusion, using digital Brix refractometer at a Brix threshold of <23% could reduce feeding of low-quality colostrum to calves and improve colostrum and calf management practices in Atlantic Canada dairy herds. The TIR spectroscopy showed high Se in detection of low-quality colostrum. However, the RID assay, which is used as the reference test in several studies, showed limited Se for detection of low-quality colostrum.

Evaluation of Brix refractometer as an on-farm tool for colostrum IgG evaluation in Italian beef and dairy cattle.

In this study it is hypothesized that there are differences between immunoglobulin G (IgG) content in colostrum from beef (Chianina, Podolica) and dairy (Holstein Friesian) cows and that variables such as breed, and parity can influence IgG content. The further objective was to determine if these factors may vary in terms of sensitivity, specificity and the cut point when data obtained with the digital Brix refractometer is compared with the gold standard radial immunodiffusion assay (RID). A total of 90 samples of first-milking colostrum were collected within 2 h after parturition. IgG concentration was determined indirectly by digital Brix refractometer and directly by RID. Results obtained by RID were compared among breed and parity. For the digital Brix refractometer, sensitivity and specificity to detect colostrum with an IgG concentration lower than 50 g/l were calculated and the optimal cut-point was selected for each breed. Samples containing less than <50 g/l IgG accounted for 15.9% of the total. Parity influenced colostral IgG concentration and beef cows had a higher mean concentration of IgG (101.1 g/l in Chianina and 90.6 g/l in Podolica) than dairy cows (71.1 g/l in Holstein Friesian) First parity Chianina cows had the highest IgG mean content (116.1 g/l). At the optimal cut-point for Brix refractometer (20%) sensitivity and specificity were 0.93 (0.84-0.97) and 0.81 (0.70-0.88), however, a breed-related cut-point could be used to reduce evaluation error. Linear regression modeling showed that refractometer data were related to RID (r = 0.78). Results obtained suggest that breed and parity can influence IgG content of colostrum and, despite the Brix refractometer being an excellent on-farm tool, a breed-based definition of optimal cut point is needed.

Determination of the potency of a cell-based seasonal quadrivalent influenza vaccine using a purified primary liquid standard.

In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine.

Using serum and plasma samples to assess failure of transfer of passive immunity in dairy calves.

The objectives of this study were (1) to determine the differences in IgG and total protein (TP) content of serum and plasma samples collected from the same calves; (2) to evaluate the correlation between calf serum and plasma IgG levels, Brix scores, and TP concentrations; (3) to determine whether different cut-off values should be used for plasma and serum to assess failure of transfer of passive immunity (FTPI) in dairy calves; and (4) to evaluate the level of agreement between results obtained from using serum and plasma samples of the same calves to assess FTPI using optimal cut-off values. Blood samples (n = 217) were collected from Holstein calves at 3 to 10 d of age on 30 commercial dairy farms in Nova Scotia and Newfoundland, Canada. Paired serum and plasma samples were analyzed for IgG concentration by the reference radial immunodiffusion assay, transmission infrared (TIR) spectroscopy, digital and optical Brix refractometers, and optical TP refractometer. The IgG concentrations measured by RID and TIR spectroscopy in serum were similar to those in plasma. However, the Brix and TP refractometer readings were significantly higher in plasma than in serum. The prevalence of FTPI in serum and plasma samples based on a RID-IgG concentration <10 g/L was 43.3 and 46.5%, respectively. The RID-IgG concentration was correlated with TIR-IgG (r = 0.92 and 0.89), digital Brix (r = 0.80 and 0.80), optical Brix (r = 0.77 and 0.77), and optical TP (r = 0.75 and 0.77) refractometers in serum and plasma, respectively. The correlations between paired serum and plasma IgG content were 0.85 by TIR spectroscopy, 0.80 by digital Brix, 0.77 by optical Brix, and 0.79 by optical TP refractometer. The optimal cut-off values for TIR spectroscopy, digital Brix, optical Brix, and TP refractometers to assess FTPI using serum were 13.1 g/L, 8.7% Brix, 8.4% Brix and 5.1 g/dL, respectively; and the optimal cut-off values with plasma were 13.4 g/L, 9.4% Brix, 9.3% Brix and 5.8 g/dL, respectively. When using these optimal cut-off values, the level of agreement (88.1%) between results derived from testing serum and plasma by TIR spectroscopy was substantial, with a kappa (κ) value of 0.76. The results derived from testing serum and plasma by digital Brix refractometer showed substantial agreement (83.4%), with a κ value of 0.65, which is higher than the agreement and κ value (74.7% and 0.51) reported for the optical Brix refractometer. Substantial agreement (81.6%) between serum and plasma TP was also obtained when using the optical TP refractometer, with a κ value of 0.63. In conclusion, serum or plasma samples can be used interchangeably for measuring IgG concentrations and assessing FTPI in dairy calves. However, different cut-offs must be used to assess FTPI depending on the sample matrix. Furthermore, results obtained from serum samples showed higher agreement with the reference RID assay than those obtained from plasma samples.

A review of diagnostic tests for diagnosing failure of transfer of passive immunity in dairy calves in New Zealand.

The aim of this review is to critically assess the test characteristics and practicality of published data on direct and indirect tests for diagnosing failure of transfer of passive immunity (FPT) in dairy calves in New Zealand, to provide recommendations for veterinary practitioners, and to examine the recommended sample size for assessing herd-level prevalence of FPT and the confidence in the results obtained. The definition of FPT is based on measurement of concentrations of IgG in serum of neonatal calves after colostrum intake. The gold standard method for measurement of concentrations of IgG is radial immunodiffusion. However its cost, requirements for laboratory equipment, and the time taken to obtain results have meant that alternative tests have been developed. The turbidimetric immunoassay and ELISA also directly measure concentrations of IgG. Indirect tests include measurement of concentrations of total proteins (TP) in the laboratory or using a refractometer, γ-glutamyl transferase (GGT) activity, and the zinc sulfate turbidity (ZST) test. Of the indirect tests, measurement of concentrations of TP in the laboratory or using a refractometer combine high specificity and sensitivity with a consistent association with concentrations of IgG in calves between 1-7 days of age. Using a refractometer is less accurate than direct measurement in a laboratory, but is still a suitable test if low cost and speed are important. Although GGT activity is strongly associated with concentrations of IgG in serum, the relationship varies with time after birth. Therefore the target thresholds change with time, increasing error compared to the measurement of concentrations of TP in serum. Similarly, factors other than total concentrations of IgG have a significant effect on the association with ZST test, complicating interpretation. Thus, when direct measurement of concentrations of IgG is not feasible, the recommendation is that concentrations of TP in serum should be used as the diagnostic test for diagnosis of FPT, providing calves are not dehydrated. Using a sample size of 12 calves is suitable for estimating whether the herd-level prevalence of FPT is <20% or >20%, if there are no calves or >5 calves diagnosed with FPT, respectively, but is limited in diagnostic confidence when 1-4 calves test positive. Diagnostic interpretation can be significantly improved if tests of FPT are used alongside information on the likely risk of FPT on the tested farm.

[Laboratory diagnostics of ovarian cancer among the elderly in the parameters of immunoglobulins and phagocytic activity of blood.]

Due to the increase in the proportion of elderly women, the prevalence of ovarian cancer is increasing, which requires the search for new methods of its diagnosis. The aim of the work is to analyze the content and informativeness of immunoglobulins and phagocytic activity of peripheral blood to improve the diagnosis of ovarian cancer among elderly women. In 78 patients with ovarian cancer aged 65-70 years immunological and spectrophotometric methods studied phagocytic activity and the level of peripheral blood immunoglobulins. 42 women of the same age with no ovarian cancer served as control. The primary importance of circulating immune complexes, phagocyte migration inhibition index and neutrophil activity index for laboratory diagnosis of ovarian cancer among the elderly was established. The use of highly informative parameters of systemic immunity improves the diagnosis of ovarian cancer in the elderly.

Analysis of the proficiency of single radial immunodiffusion assays for quality control of influenza vaccines in Korea.

Influenza vaccine potency, which is determined by quantitatively measuring the content of Hemagglutinin (HA), is an essential index representing the efficacy of the vaccine. Standardization of the single radial immunodiffusion (SRID) assay, a method for measuring HA content, and proficiency of the testing institutions are crucial for influenza vaccine quality control. Herein, we assessed the proficiency of SRID assays at the National Control Laboratory (NCL) of Korea and several vaccine manufacturers. Eight laboratories participated in this study, and the proficiencies of all laboratories yielded satisfactory results in overall SRID assays. In contrast, there were some unsatisfactory results in measuring with different types of agarose gel plates produced by other laboratories. Overall, our findings demonstrated that the proficiency of SRID assay in the tested laboratories is acceptable for quality control of influenza vaccines and that detailed review on the validation reports regarding the test methods will be helpful for better control.

Colostrum immunoglobulin G concentration of multiparous Jersey cows at first and second milking is associated with parity, colostrum yield, and time of first milking, and can be estimated with Brix refractometry.

The objective of this study was to evaluate colostrum IgG concentration harvested at first and second milking from multiparous Jersey cows, the dam's lactation number, colostrum yield, and time of first milking. In addition, we validated the use of a Brix refractometer to estimate IgG concentration in colostrum from multiparous Jersey cows using radial immunodiffusion as the reference method. Colostrum samples and total weight of colostrum harvested at first (n = 134) and second (n = 68) milking were collected from 134 multiparous Jersey cows housed in a California herd. Fresh colostrum samples were analyzed for IgG concentration with Brix refractometry and frozen samples by radial immunodiffusion. A total of 90.4 and 42.7% of the samples from first and second milking met industry standards of quality for IgG concentration (>50 g/L). Second and third lactation cows had similar colostrum IgG concentration but lower than cows on their fourth and greater lactation. At second milking, 56.4% of cows on their fourth or greater lactation had colostrum IgG concentrations >50 g/L. When colostrum yield increased from low (<3 kg), medium (3 to 6 kg), to high (>6 kg), IgG concentration decreased. Higher IgG concentration was observed on colostrum harvested at <6 h (short) versus 6 to 11 h (medium) after calving. However, IgG concentration in colostrum harvested after 11 h (long) was similar to that harvested at short and medium time. Readings of %Brix were highly correlated with IgG at first (r = 0.81) and second (r = 0.77) milking. The best Brix threshold to identify colostrum from first milking with >50 IgG g/L was 20.9% based on logit equations with Youden's index criterion and 18.0% based on accuracy criterion. For colostrum harvested at second milking, similar Brix thresholds were obtained, 19.2 and 19.0%, regardless of whether Youden's index or accuracy was used as the selection criterion. Our results indicate that the dam's lactation number, colostrum yield, and time of first milking relative to calving are associated with IgG concentration in colostrum from multiparous Jersey cows. Second milking colostrum from mature Jersey cows should be evaluated to extend colostrum supply on dairies especially during times of shortage. Readings of %Brix can be used to rapidly estimate IgG concentration in Jersey colostrum harvested at first and second milking.

Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum.

Capillary electrophoresis (CE) is a technique routinely used in clinical laboratories that allows the separation and quantification of blood serum proteins in a rapid, precise, accurate, and inexpensive manner. Recently, CE has been proposed to separate and measure colostral proteins, but an evaluation of the agreement between CE and radial immunodiffusion (RID) method, currently used to quantify IgG in colostrum, is still lacking. The purpose of this study was to test the ability of a CE instrument, normally used in blood serum protein analysis, to realize the correct quantification of total Ig concentration in ewe colostrum, using RID assay as reference. Colostrum samples (n = 68) were collected from 35 multiparous Sarda ewes at first milking (n = 33) and at 24 h postpartum (n = 35). The mean ± standard deviation of IgG concentration measured by RID and whey colostrum total Ig concentration measured by CE were 54.76 ± 41.82 g/L and 54.70 ± 41.43 g/L, respectively. Lin's concordance correlation coefficient (r = 0.993; 95% confidence interval = 0.989 to 0.996) and linear regression analysis results (RID = 1.0022CE - 0.063; R2 = 0.986) showed an excellent agreement between these 2 methods. Bland-Altman analysis confirmed that CE method can be a suitable alternative to RID: the mean of the differences between CE and RID was -0.055 ± 4.95 g/L (95% confidence interval = -1.25 to 1.14 g/L) and the agreement limits were -9.75 to 9.60 g/L (low limit 95% confidence interval = -11.82 to -7.68 g/L; high limit 95% confidence interval = 7.57 to 11.72 g/L). In conclusion, the current study indicates that CE method may be a reliable tool for the quantification of the total Ig concentration in ewe colostrum.

Rapid assessment of bovine colostrum quality: How reliable are transmission infrared spectroscopy and digital and optical refractometers?

The objectives of this study were to evaluate the performance of the transmission infrared (IR) spectroscopic method and digital and optical Brix refractometers for measurement of colostral IgG concentration and assessment of colostrum quality of dairy cows. Colostrum samples (n = 258) were collected from Holstein cows on 30 commercial dairy farms in Nova Scotia and Newfoundland, Canada. Colostral IgG concentrations of 255 samples were measured by the reference radial immunodiffusion (RID) assay and IR spectroscopy. The Brix scores were determined on 240 of these samples using both the digital and optical Brix refractometers. Approximately half (48%) of the colostrum samples had RID IgG concentrations <50 g/L, which was the cut-point for poor quality. The correlation between RID and IR IgG concentrations was 0.88. The correlations between RID IgG concentration and Brix scores, as determined by the digital and optical refractometers, were 0.72 and 0.71, respectively. The optimal cutoff levels for distinguishing good- and poor-quality colostrum using IR spectroscopy, and digital and optical Brix refractometers were at 35 g/L and 23% Brix, respectively. The IR spectroscopy showed higher sensitivity (90%) and specificity (86%) than the digital (74 and 80%, respectively) and optical (73 and 80%, respectively) Brix refractometers for assessment of colostrum quality, as compared with RID. In conclusion, the transmission-IR spectroscopy is a rapid and accurate method for assessing colostrum quality, but is a laboratory-based method, whereas Brix refractometers were less accurate but could be used on-farm.

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.

Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.

Technical note: Comparison of radial immunodiffusion and ELISA for quantification of bovine immunoglobulin G in colostrum and plasma.

Historically, radial immunodiffusion (RID) has been the only method that directly measures IgG; however, recent studies have reported IgG concentrations in colostrum, milk, and plasma as measured using an ELISA. To our knowledge no comparison between RID and ELISA methods has been made for bovine colostrum or plasma. The objective of this study was to compare IgG concentrations measured by both methods in samples of bovine colostrum before and after heat treatment and bovine plasma. Concentration of IgG was quantified using a commercially available RID kit and a modified ELISA. Samples of bovine colostrum and plasma were collected from individual animals and colostrum was tested before and after heat treatment at 60°C for 30 min. All samples were tested using both methods. Pearson correlation coefficients were determined for RID and ELISA values from unheated colostrum, heat-treated colostrum, and plasma samples. Mixed models were used to determine the effect of assay on IgG measurement in colostrum and plasma and effect of heat treatment on IgG concentration in colostrum. A weak correlation was found between ELISA and RID results in plasma and unheated colostrum. Concentration of IgG was significantly lower in all sample types when measured by ELISA compared to RID. Thus, direct comparison of ELISA and RID results is not recommended. Colostrum IgG concentration significantly decreased after heat treatment as measured by ELISA, but means were not different when measured by RID. Correlation plots between colostrum values measured before and after heat treatment indicated changes in the colostrum protein matrix due to heat affected RID and ELISA assays differently. This investigation compared RID and ELISA results, but no conclusions could be drawn as to the accuracy of either assay.

Evaluation of on-farm tools for colostrum quality measurement.

The objectives of this study were to determine the immunoglobulin G (IgG) content of colostrum on Alberta dairy farms and to determine which on-farm tool, the colostrometer or the Brix refractometer, was more highly correlated with IgG content as determined by radial immunodiffusion (RID). Colostrum samples (n=569) were collected between February and July 2012 from 13 commercial dairy farms in central Alberta, with herds ranging in size from 60 to 300 lactating cows. Immunoglobulin G content was determined directly by RID and indirectly by a colostrometer (specific gravity) and Brix refractometer (total solids). The Spearman correlation was used for the colostrometer and Brix refractometer data. According to RID analysis, 29.1% of the colostrum samples contained <50 mg/mL IgG. Concentrations ranged from 8.3 to 128.6 mg/mL IgG, with a median of 65.1 mg/mL. Third or greater parity cows had higher colostral IgG content (69.5±1.98 mg/mL) than second parity (59.80±2.06 mg/mL) or first parity (62.2±1.73 mg/mL) cows. The colostrometer data were more highly correlated with RID results (r=0.77) than were the Brix refractometer data (r=0.64). Specificity and sensitivity were determined for the colostrometer and Brix refractometer compared with a cut-point of 50 mg/mL IgG as determined by RID. The highest combined value for sensitivity and specificity occurred at 80 mg/mL for the colostrometer (84.1 and 77.0%, respectively) and 23% Brix (65.7 and 82.8%, respectively). This study indicates that although the colostrometer data are better correlated with true IgG values, the user-friendly Brix refractometer is a more specific tool to detect colostrum of adequate quality.

Preliminary validation of a calf-side test for diagnosis of failure of transfer of passive immunity in dairy calves.

The objective of this study was to evaluate the utility of an initial version of a calf-side test (ZAPvet Bovine IgG test, ZBx Corp., Toronto, ON, Canada) for diagnosis of failure of transfer of passive immunity (FTPI) in dairy calves. Blood samples (n=202) were collected from calves from 1 to 11d of age. Serum IgG concentration was determined by radial immunodiffusion (RID) assay. The mean IgG concentration was 1,764±1,035mg/dL, with a range from 133 to 5,995mg/dL. The ZAPvet Bovine IgG test was used to assess FTPI (serum IgG <1,000mg/dL) and test characteristics were calculated. The number of samples that had FTPI from the RID assay and ZAPvet test was 55 and 96 samples, resulting in a true prevalence of 27% and an apparent prevalence of 47.5%, respectively. The sensitivity, specificity, and positive and negative predictive values of the ZAPvet test were 0.82, 0.65, 0.47, and 0.91, respectively. The results of the ZAPvet test were derived from 2 observers, and the overall level of agreement between the results of the 2 observers was 84%, with a kappa value of 0.67. The ZAPvet Bovine IgG test showed good potential for further development as a cost-effective, rapid calf-side test for monitoring FTPI in dairy calves.

Immunity transfer in mule foals fed with good IgG quality colostrum.

While the passive transfer of immunity in horse and donkey foals has been extensively studied, there is limited information for mule foals. Immunoglobulin type G (IgG) and serum total protein concentration (TP) were assessed at different sampling times to evaluate the correlation between serum radial immunodiffusion (SRID) with electrophoresis, refractometry, and dry chemistry analyzer (Biuret), and to estimate serum IgG concentrations using serum TP in mule foals. We analyzed a total of 30 samples collected at birth, and at 6, 12, 24, and 48 h of life from 6 mule foals by SRID, electrophoresis TP, biuret TP, and refractometry TP. The SRID IgG concentration significantly increased from birth until T6 (p < 0.001). Serum TP analyzed with refractometry revealed differences between T0 and T12, T24 and T48 (p < 0.05), while a significant difference was observed with the biuret method between T0 and all the other sampling times (p < 0.001). A strong correlation was found between IgG SRID and biuret TP (r = 0.69, p < 0.001), and a good correlation existed between IgG SRID, refractometry TP, and electrophoresis TP (r = 0.44, p < 0.01 and r = 0.39, p < 0.05, respectively). All methods can be used to estimate the passive transfer of immunity in mule foals. TP refractometry and biuret TP values can be used to determine serum IgG concentrations in the blood of mule foals on their first day of life through the application of a specific equation.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Investigation of brix refractometry for estimating bovine colostrum immunoglobulin G concentration.

Many dairy operations uses a Brix refractometer to assess the quality of first-milking colostrum. This study investigated whether a digital Brix refractometer could be used in a model to predict colostrum IgG concentration and whether more than one %Brix threshold could be used for different colostrum IgG concentrations. Colostrum from 182 animals was tested using a digital Brix refractometer and by single radial immunodiffusion. Statistical analysis, using simple linear regression to relate %Brix results with corresponding colostral IgG concentration, and receiver operating characteristic (ROC) analysis were used to identify %Brix cutoffs that had no false positive results. Colostral IgG concentrations from digital Brix refractometry had a R2 value of 0.818 and a S-value of 21.7 g/L. The large S-value shows that a digital Brix refractometer should not be used in a model to predict colostrum IgG concentration. However, %Brix scores of 19.0, 22.0, 25.0 and 30.0 percent can be used to estimate minimum colostral IgG concentrations of 25, 50, 75 and 100 g/L. These four cutoffs can be used to strategically feed smaller volumes of colostrum to newborn calves. Smaller volumes may reduce unwanted side effects and shorten the time interval in which calves refuse to nurse, while still delivering an adequate mass of IgG to have successful transfer of passive immunity.