Radial Immunodiffusion Assay Research Articles

A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts.

In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.

Assessing colostral and serum immunoglobulin G in alpacas using Brix refractometry and total serum protein

The adequate transfer of passive immunity is a critical factor in neonatal development and survivability. Although well documented in the dairy and equine industries, the recognition of inadequate immunoglobulin transfer on-farm and its impact on the ability of alpaca cria to thrive is largely unknown. Colostrum samples were collected from female alpaca within 24 h of parturition by the owners and whole blood collected from cria by the investigators between 1 and 7 days of age. Direct IgG concentration of milk and serum was determined using radial immunodiffusion assay (RID) and was indirectly estimated using optical and digital Brix refractometry for total solids and clinical refractometry for total serum protein. There was a strong correlation between optical and digital Brix refractometry, and colostral IgG concentration determined by RID. There was a moderate correlation between serum IgG concentration determined by RID and total serum protein in crias. Optical and digital Brix refractometry for colostral IgG estimation and total serum protein for serum IgG estimation are reliable, accurate and easy-to-use tools that can be used on-farm by trained, competent technicians to assess a failure of passive transfer in alpacas. A pilot study at one property only was performed, due to COVID-19 travel restriction interference. Further research is required to determine the reference intervals for these tools to be practical.

PSIV-1 Effects of Poor Maternal Nutrition During Gestation on F0 and F1 Ewe Colostrum and Milk Composition and Colostrum Igg

Abstract High-quality colostrum and milk are critical for proper neonatal growth and immune system development. Immunoglobulin G (IgG) is the predominant antibody class in colostrum and is vital for passive immune transfer in sheep. There is limited understanding of how poor maternal nutrition during gestation (restricted- and over-feeding) might influence colostrum and milk composition and colostrum IgG concentrations and how F1 offspring colostrum and milk are impacted as a result of F0 maternal diet. The objective of this experiment was to determine if poor maternal nutrition during gestation impacts F0 and F1 colostrum and milk composition, including IgG concentrations. We hypothesized that maternal restricted-feeding would decrease, and maternal over-feeding would increase total solids, fat, and protein during early and transitional lactation and that restricted-feeding would increase IgG concentrations and over-feeding would decrease IgG concentrations in F0 and F1 ewe colostrum. Multiparous Dorset ewes pregnant (n = 46) with twins were fed 100% (control; n = 13), 60% (restricted; n = 17) or 140% (over; n = 16) of National Research Council (NRC) requirements. After birth and during lactation, dams were fed 100% NRC for lactating ewes, so the nutritional insult only occurred during gestation. Resulting ewe offspring (n = 32) were raised on control diets and were bred between 16 and 19 mo of age. Colostrum samples were collected within 24 h of parturition and milk samples were collected at d 3 and d 21 postpartum. Colostrum and milk composition were evaluated using Brix Refractometry (total solids, g/dL) and spectrophotometry (crude fat, %; crude protein, %). Additionally, a radial immunodiffusion assay was used to determine IgG concentrations (mg/dL) in colostrum. A treatment by time interaction was observed for F0 colostrum crude fat where restricted-fed ewes had less fat than control-fed ewes (P ≤ 0.024). No interaction was detected for all other F0 and F1 composition variables (P ≥ 0.16). We did not detect a main effect of diet on F0 or F1 total solids, crude fat, and crude protein of colostrum nor milk (P ≥ 0.24). A main effect of time was observed for all variables where colostrum had greater total solids, crude fat, and crude protein compared with milk at d 3 and d 21 (P ≤ 0.0001). No effect of maternal diet on IgG concentrations were detected in F0 or F1 ewe colostrum (P ≥ 0.46). These data indicate that poor maternal diet has minimal effects on F0 and F1 ewe colostrum composition and IgG concentrations, and d 3 and d 21 milk composition. In addition, the impact of F0 diet on colostrum fat composition did not persist into the F1 generation.

Evaluation of an immunochromatographic point-of-care test for the detection of failure of transfer of passive immunity in calves

BackgroundAs calves are born without circulating immunoglobulin G (IgG) they depend on transfer of passive immunity via colostrum within the first hours of life. If calves are not sufficiently supplied with high qualitative colostrum they suffer from Failure of Transfer of Passive Immunity (FTPI). The objectives of this study were to evaluate a calf-side point-of-care test to detect calves with FTPI and to evaluate the cut-offs for a positive test result. Two hundred fifty calves from 11 dairy farms (born between September 2021 and September 2022) were included, whereof 23 were excluded due to incomplete data. Twelve to 16 h post partum the farmers carried out a point-of-care test (FASTest® IgG bovine, Megacor, Austria) using a whole blood sample. Between the 3rd and the 6th day of age, all calves were physically examined and blood samples were collected to carry out further point-of-care tests using whole blood supernatant and plasma and for measuring the Brix values in serum and plasma. Brix values in serum were used as reference for the evaluation of the point-of-care test between the 3rd and the 6th day of age, as radial immunodiffusion assays could not be conducted simultaneously.ResultsBrix values were not normally distributed (median at 8.6% and 9.3% in serum and plasma). In this study, the cut-off values for the point-of-care tests using whole blood supernatant and plasma were at 8.3% Brix in serum. FASTest® IgG bovine shows high sensitivities of 90% and 84% and specificities of 70% and 72% for whole blood supernatant and plasma.ConclusionsOf the 227 investigated calves, 39.7% showed Brix values of < 8.4% (cut-off for FTPI) which indicates an urgent need to improve colostrum management. The results of the study suggest that the FASTest® IgG bovine is a suitable on-farm method to assess FTPI in whole blood supernatant and plasma of calves between the 3rd and the 6th day of age. However, the results also show that FASTest® IgG bovine is not adequate to test for FTPI using whole blood at 12 to 16 h post partum.

Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Transfer of passive immunity and survival in Jersey heifer calves fed heat-treated pooled colostrum.

Acquisition of adequate transfer of passive immunity (ATPI) by calves depends on the absorption of sufficient mass of colostral immunoglobulin G (IgG). Several studies report conflicting evidence regarding the ability of feeding pooled colostrum to achieve ATPI. Pooling colostrum is practical and efficient for some dairies, and recommendations are required to prevent failure of transfer of passive immunity (FTPI) in calves following pooling. This study aimed to determine the effect of pooling colostrum on serum IgG concentrations, FTPI, and preweaning mortality in calves. A prospective study was performed on two conventional Jersey dairy farms where heat treatment of colostrum occurred in the same colostrum processing kitchen. Four to 10 cows contributed to colostrum pools. A sample of the colostrum pool fed to the calves and serum from calves at 24-72 h was collected for IgG concentration determination by single radial immunodiffusion assay. Multivariable and logistic regression analyses were performed to evaluate factors that predicated serum IgG concentrations and the probability of FTPI, respectively. A Cox proportional hazard model analysis was performed to determine risk factors for mortality over the preweaning period. A total of 164 calves fed 28 colostrum pools were enrolled. Birth weight, number of colostrum feedings and pool IgG concentrations were significant predictors of calf serum IgG concentrations at 24-72 h, whereas the number of colostrum feedings and age at bleeding to determine passive transfer status were not significant predictors of calf serum IgG concentrations at 24-72 h. The prevalence of FTPI was 4.9%. Birth weight, pool IgG concentrations, number of colostrum feedings, and age at bleeding to check for passive transfer status were not significant predictors of the probability of FTPI at 24-72 h. The incidence of mortality was 4.3%. Passive transfer status was not a predictor of mortality. Our study demonstrates the effect of pooling colostrum on serum IgG concentrations even in herds where colostrum with higher median colostrum IgG concentrations is fed to calves. The results emphasize the recommendations to assess pooled colostrum IgG concentrations before feeding calves.

Association of a green tea extract with serum immunoglobulin G status and neonatal vitality in newborn dairy calves.

Poor vigor at birth has been associated with reduced IgG absorption from colostrum and a reduced vitality in neonatal dairy calves. Some natural compounds, such as green tea extract, may improve vitality in compromised calves. The objective of this randomized controlled trial was to evaluate the potential of supplementing a green tea extract (15 mL) to calves to improve vigor and activity behavior for the first 72 h postnatal. Also, this study aimed to investigate the influence of green tea extract supplementation on calf serum IgG concentration and the apparent efficiency of absorption (AEA) of colostral IgG. Holstein calves (n = 24) weighing 42.49 ± 1.07 kg postnatal received a complete random assignment at 3 h of one 15-mL dose of green tea extract (Calf Perk, TechMix) or distilled water orally before tube feeding colostrum replacer (Premolac Plus IgG, Zinpro) at 4 h postnatal. Two observers assessed for calving time and dystocia by live video stream to retrieve all calves within 2 h postnatal. One veterinarian performed a baseline vigor assessment based on heart rate and response to stimuli on all calves at 2.5 h, before colostrum feeding at 3.75 h, as well as at 24, 48, and 72 h postnatal. Calf blood samples were taken to assess total IgG by radial immunodiffusion assay at 2.5, 6, 12, 24, 48, and 72 h. Calf vitality was also observed continuously by video for all calves to determine whether treatment was associated with attempts to stand, lying time, and exploration of their pen environment for the first 24 h. We used an ordinal logistic model to evaluate the odds of green tea extract improving a calf's vigor category from 2.5 h postnatal to 72 h of age. Vigor score was categorized as abnormal (≤4), average (5), or alert (≥6), with hour as a fixed effect. We also ran mixed linear models to evaluate the effect of extract on total IgG and AEA, with time and dystocia as fixed effects. Five dystocia calves were enrolled (2 control, 3 extract), but assistance was minor (e.g., manual assistance and all were assisted within 1 h). Baseline vigor scores and baseline total IgG were not different between groups. Vigor score category was not associated with green tea extract supplementation (odds ratio 1.17; 95% CI: 0.43-3.15) but increased with time compared with controls. We observed no association of treatment with total IgG or AEA in the calves, suggesting green tea extract does not compromise IgG absorption. Calf vitality, lying behavior, and exploratory behavior were not associated with green tea extract treatment. Our findings suggest that green tea extract supplementation does not affect AEA and serum IgG concentration in calves. Future research should evaluate whether green tea extract improves vitality in calves experiencing severe dystocia.

Validation of a handheld refractometer to assess Merino ewe colostrum and transition milk quality

Colostrum quality is generally defined by the IgG concentration in colostrum, and many methods have been used to assess it. Methods to measure colostrum quality both in the laboratory and in the field have been validated in cattle; however, this is only a recent topic of interest for sheep colostrum. Laboratory-based methods are often time consuming and require trained personnel compared with new handheld evaluation tools such as the digital Brix refractometer, which gives real-time results. The aims of this study were to (1) evaluate the relationship between the digital Brix refractometer and constituents indicative of quality (IgG, protein, fat, and lactose) in colostrum and transition milk, and (2) determine an appropriate Brix % cut-off value for the Brix refractometer in sheep colostrum and transition milk. The study used 50 colostrum samples (collected at 0 h postpartum, before lambs' sucking) and 169 transitional milk samples (collected at 4 and 24 h postpartum, after lambs had sucked) collected over 6 lambing trials in 2 years (2019 and 2020). We concluded that the Brix refractometer results correlated weakly with IgG concentration determined by radial immunodiffusion assay in colostrum collected at 0 h postpartum (r = 0.11) and in transition milk collected at 4 h postpartum (r = 0.12); however, a moderate to strong correlation was shown in transition milk samples collected at 24 h (r = 0.66). Brix % was significantly correlated with fat %, lactose %, and protein % at all timepoints. To determine an appropriate Brix % cut-off value indicating an IgG concentration of 20 mg/mL, we analyzed sensitivity and specificity of the Brix refractometer at 0, 4, and 24 h. In samples collected at 0 and 4 h, the highest combination of sensitivity, specificity, and accuracy was achieved at a Brix % cut-off value of 29%; in samples collected at 24 h postpartum, a Brix % cut-off value of 27% gave the highest sensitivity, specificity, and accuracy. Overall, the Brix refractometer has potential as a useful in-field tool for researchers and producers in both extensively and intensively managed flocks to measure and determine the quality of sheep colostrum and transition milk.

Changes in serum total protein and immunoglobulin G concentrations and Brix percentages in neonatal Arabian foals from birth up to 21 days of age

The aim of this study was to evaluate changes in the serum immunoglobulin G (IgG) and serum total protein (STP) concentrations and serum Brix percentages of neonatal Arabian foals during first 3 weeks of life. Blood samples were collected from 12 apparently healthy foals by jugular venipuncture at birth and at 12-hours, 24-hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 21 days of age. Serum IgG and STP concentrations and Brix percentages were measured by the radial immunodiffusion assay, and digital STP and Brix refractometers, respectively. Based on the serum IgG concentrations measured at 24 h, two foals were diagnosed with failure of transfer of passive immunity (FTPI). While IgG concentrations were determined using the data of foals with adequate transfer of passive immunity, other calculations were made using the data of all foals. The mean IgG concentration of the foals increased from birth (<200 mg/dl) to 12 (2068.5 mg/dl) and 24 h (2184.7 mg/dl), and progressively decreased up to 21 days of age (1318.5 mg/dl). The serum IgG concentrations at 12 h were highly correlated with each of the IgG concentrations measured over the 21-day period. The serum IgG and STP concentrations and Brix percentages of the foals diagnosed with FTPI at 12 h did not reach the adequate strata over time. These results suggest that foals can be reliably tested for passive immunity status at 12 h after birth.

Sources of variance in the results of a commercial bovine immunoglobulin G radial immunodiffusion assay.

Radial immunodiffusion (RID) is used to quantify IgG concentration in neonatal beef or dairy calf serum; variability has been noted that may affect the precision and accuracy of assay results. We determined the source, range, and homogeneity of variance in the results of a commercial bovine IgG RID assay (Triple J Farm). To estimate the variance in the precipitin ring diameter, we used 6 sera, measured 28 times across 8 plates and 4 lots, and 3 standards with known IgG concentrations, measured 75 times across 69 plates and 5 lots. The source of diameter variance was determined using variance partition coefficients for lot, plate, and repetition. We used 11 different methods to generate standard curves to convert RID precipitin ring diameters to IgG concentrations. The Levene test of homogeneity of variance (α = 0.1) was used to evaluate the equality of variance between the standards or serum precipitin ring diameters and calculated IgG concentrations. Lot and plate contributed minimally to the diameter variance. Precipitin ring diameters had equal variance. Calculated IgG concentrations for serum not requiring dilution had equal variance. A linear equation from aggregated standards, performed within the same day, had greater accuracy for the calculated IgG concentrations of the standards compared to other equation methods. Regardless of standard curve methodology or IgG concentration, variability inherent to the assay limits its clinical usefulness.

Physicochemical and antigenic properties of the urea-extracted surface structures of &lt;i&gt;Yersinia pseudotuberculosis&lt;/i&gt; O:1b

Immuno-serological diagnostic tools particularly identifying pathogen antigens are the most important methods of pseudotuberculosis studies. The main immunodominant and species-specific antigens located in the surface structures of the bacterial cell are of practical interest. Thereby the aim of the work was to isolate and characterize biologically active surface structures of the pseudotuberculosis microbe. Here, the living cells of Y. pseudotuberculosis 3704 (O:1b) were lysed by using 9 M urea solution to extract antigens localized in the microbial surface structures. The subcellular fractions obtained such as outer membranes (OM), urea extract (UE) and isolated protein-lipopolysaccharide complex (PLPSC) are characterized by physical and chemical parameters. The protein content in the preparations ranged from 42% to 53%. The polypeptide band of the OM preparation, UE polypeptide and PLPSC for pseudotuberculosis microbe was presented by 14, 16 and 9 major polypeptides with molecular weight ranging from 13.9 kDa to 131.5 kDa, 13.5 kDa to 101.6 kDa, and 20.7 kDa to 66.6 kDa, respectively. Proteolytically active proteins and polypeptides were detected in isolated subcellular fractions (OM and UE) by using the radial enzyme diffusion test and substrate-gel electrophoresis found to be presented by 4 and 7 polypeptides with molecular weight ranging from 28.0 kDa to 118.0 kDa and 29.2 kDa to 97.7 kDa in the OM and UE preparation, respectively. The subcellular fractions obtained are capable to exhibit immunogenic activity after inoculation to experimental animals and antigenic activity while interacting with specific antibodies in the radial immunodiffusion (RID) assay and antibodies labeled with colloidal silver nanoparticles in dot immunoassay (DIA). OM and PLPSC preparations in DIA with immunoglobulins isolated from experimental antisera and labeled with colloidal silver nanoparticles were detected at a concentration of 0.12 g/ml (dry weight), cells of strain Y. pseudotuberculosis 3704 at a concentration of 3, 9 106 m.c./ml, which is similar to the results of DIA with immunoglobulins isolated from commercial pseudotuberculosis antiserum (St. Petersburg) and labeled with nanoparticles of colloidal silver. Thus, the subcellular fractions of pseudotuberculosis microbe isolated by using urea as a lysing and decontaminating agent retain their antigenic and immunogenic properties and enzymatic activity suggesting about their potential benefits for use to improve early diagnostics of pseudotuberculosis.

Diagnostic accuracy of refractometry methods for estimating passive immunity status in neonatal beef calves.

Assessing the inadequate transfer of passive immunity (ITPI) in beef calves is crucial because calves with ITPI are at high risk for morbidity and mortality. The aim of this study was to determine the accuracy of digital Brix (D-BRIX) and digital serum total protein (D-STP) refractometers to estimate different passive immunity status in beef calves and to determine the robustness of thresholds. Blood samples were collected from 202 (1-7 days old) beef calves. Serum total solid percentages, total protein concentrations, and IgG concentrations were measured with the D-BRIX refractometer, D-STP refractometer, and gold standard radial immunodiffusion (RID) assay, respectively. Data were analyzed using diagnostic test accuracy, areas under the receiver operating characteristics curve, Cohen's kappa coefficient, and misclassification costs analysis to estimate IgG concentrations <10, <16, and <24 mg/mL. For the prediction of serum IgG concentrations <10, <16 and <24 mg/mL, the optimal cut-off values were determined to be <8.5% (Se: 100.0% (95% CI: 87.9-100.0); Sp: 94.2% [95% CI: 89.6-97.2]), <8.5% (Se: 92.1% [95% CI: 78.6-98.2]; Sp: 97.6% [95% CI: 93.9-99.3]), and <10.1% (Se: 88.8% [95% CI: 79.7-94.7]; Sp: 67.2% [95% CI: 58.1-75.4]), respectively, for the D-BRIX refractometer; and <5.2g/dL (Se: 100.0% [95% CI: 87.9-100.0]; Sp: 93.6% [95% CI: 88.9-96.8]), <5.2g/dL (Se: 92.1% [95% CI: 78.6-98.2]; Sp: 97.0% [95% CI: 93.0-99.0]), and <6.4g/dL (Se: 87.5% [95% CI: 78.2-93.8]; Sp: 69.7% [95% CI: 60.7-77.7]), respectively, for the D-STP refractometer. The digital Brix and digital serum total protein refractometers can be used as monitoring tools for assessing passive immunity transfer in neonatal beef calves.

Evaluation of 2 refractometers to estimate different passive immunity status in Simmental dairy calves

Inadequate transfer of passive immunity (ITPI) is major risk for mortality, morbidity and decreased growth performance in dairy and beef calves. Refractometry method is used in estimating ITPI in dairy and beef calves but studies evaluating refractometers for Simmental dairy calves are limited. The objectives of this study were determining the accuracy of digital serum total protein refractometer (STP-REF) and digital Brix refractometer (BRIX-REF) in the estimating of different passive immunity status (<10, <18, <25 and <32 g/L) in Simmental dairy calves. Serum samples were collected from apparently healthy Simmental dairy calves aged 1–8 days (n = 291). Serum total solid percentages were measured using BRIX-REF, serum total protein concentrations were measured using STP-REF and serum IgG concentrations were measured using radial immunodiffusion (RID) assay as a reference test. Correlation coefficients were calculated between RID test and refractometers results, and each other. The diagnostic test performance of the refractometers at IgG concentrations of < 10, < 18, < 25 and < 32 g/L was measured by performing a receiver operating characteristics (ROC) analysis. The optimal thresholds of the refractometers were determined based on Youden's J statistics and after accounting for different costs between false positive and false negative cases using misclassification cost term analysis. The overall test performance and the agreement of the refractometers were assessed using area under the ROC curve and Cohen's kappa analysis. Optimal thresholds were determined as < 7.9, < 8.3, < 8.7, < 9.4% for the BRIX-REF, and < 4.6, < 5.2, < 5.4, < 5.8 g/dL for the STP-REF in estimating IgG concentrations of < 10, < 18, < 25, < 32 g/L, respectively. Se and Sp of BRIX-REF were 88.8% and 89.1% for < 7.9% Brix, 81.6% and 94.2% for < 8.3% Brix, 77.9% and 97.4% for < 8.7% Brix and 81.7% and 91.2% for < 9.4% Brix, respectively. Se and Sp of STP-REF 92.5% and 88.2% for < 4.6 g/dL; 92.1% and 87.1% for < 5.2 g/dL; 81.7% and 93.6% for < 5.4 g/dL; 79.0% and 94.1% for < 5.8 g/dL, respectively. Both, digital Brix and STP refractometers performed well to estimate the status of passive immunity in dairy Simmental calves.

Evaluation of refractometry methods for estimating passive immunity status in neonatal foals

The objective of this cross-sectional study was to determine the accuracy of the digital Brix and serum total protein (TP) refractometers for estimating different passive immunity status in neonatal foals. In total, 18- to 40-h old purebred Arabian foals (n=185) were used. Serum TP concentrations, total solid percentages and IgG concentrations were measured with a digital serum TP refractometer, digital Brix refractometer and the gold standard radial immunodiffusion (RID) assay, respectively. Correlation coefficients were calculated between the refractometer and RID assay results. A receiver operating characteristic analysis was used to select the optimal cut-offs for both refractometers. Test performance and agreement were evaluated using diagnostic test characteristics at optimal thresholds and areas under the ROC curve, and by calculating Cohen's kappa coefficient. The sensitivity and specificity of the digital Brix refractometer at optimal cut-offs (≤7.8%, ≤7.9%, ≤8.2%, ≤8.3%, ≤9.0%) were 100 and 69.3%; 100% and 68.5%; 70.5% and 71.0%; 88.3% and 85.5%; 88.1% and 76% to estimate RID-IgG of <400mg/dL, <800mg/dL, <1500mg/dL, <2500mg/dL and<3000mg/dL, respectively. The sensitivity and specificity of the digital serum TP refractometer at optimal cut-off (≤4.6g/dL, ≤4.6g/dL, ≤4.8g/dL, ≤5.0g/dL, ≤5.4g/dL) were 100 and 69.3%; 100% and 72.8%; 90% and 72.8%; 72.9% and 83.9%; 84.4% and 88% to estimate RID-IgG of <400mg/dL, <800mg/dL, <1500mg/dL, <2500mg/dL and<3000mg/dL, respectively. In conclusion, the refractometers showed a good potential as screening tools for the estimation of different IgG concentrations in neonatal foals.

The production and influence of anti-Vibrio parahaemolytics IgY against experimental infection of V. parahaemolyticus in cultured Fenneropenaeus indicus

The increasing number of antibiotic-resistant bacteria emphasizes the need to find alternatives to complement antibiotics. Immunotherapy may also be used as a complementary treatment against pathogens that are difficult to treat with traditional antibiotics. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. In the present study, pathogenic Vibrio parahaemolyticus was isolated from infected shrimp and studied their virulence factors including LD50 (by challenging with Fenneropenaeus indicus), proteolytic and hemolytic activities. The edible antibody IgY was raised by injecting the antigen of Extra Cellular Products (ECP) of V. parahaemolyticus to Gallus gallus domesticus during layoff period with and without the herbal immunoadjuvants, Asparagus racemosus and Glycine max (V.p wo: V. parahaemolyticus ECP without adjuvant; V.p A: V. parahaemolyticus ECP with A. racemosus and V.p G: V. parahaemolyticus ECP with G. max). Eggs were collected after five weeks of immunization and anti- V. parahaemolyticus IgY was extracted and purified. Physicochemical properties of the immunized Chickens' serum and anti- V. parahaemolyticus IgY's cross reactivity, growth inhibition assay, single radial immunodiffusion assay and bacterial agglutination were studied. The results revealed that, the serum protein parameters were significantly (P ≤ 0.001) increased in experimental groups from control group. The antibody raised with immunoadjuvants had significantly (P ≤ 0.001) higher cross reactivity, growth inhibition, single radial immunoassay and bacterial agglutination when compared with and without immunoadjuvant and control groups. Further the control and experimental anti-V. parahaemolytics IgY coated artificial diets were fed to F. indicus for 60 days. After 30 and 60 dpv (days of post vaccination), shrimps from each groups were challenged with virulent V. parahaemolyticus and studied the survival, haematological and immunological parameters. The IgY coated diets (V. p A and V.p G) fed shrimps had decreased cumulative mortality, significantly (P ≤ 0.001) improved coagulase activity, total haemocyte count and oxyhaemocyanin. The immunological parameters such as prophenoloxidase, intracellular anion production, lysozyme production and phagocytosis also improved significantly (P ≤ 0.001) in IgY treated shrimps.

Bayesian latent class analysis of the characteristics of diagnostic tests to assess the passive immunity transfer status in neonatal Belgian Blue beef calves

The aim of the current study was to assess the diagnostic characteristics of radial immunodiffusion (RID), capillary electrophoresis (CE) and digital brix refractometry (Bx) for the diagnosis of failure of passive transfer (FPT) of immunity in neonatal Belgian Blue beef calves in the absence of a gold standard using a Bayesian latent class model. Belgian blue beef calves (n = 202) from a large farm in the south of Belgium were blood-sampled at 48–72 h of age and tested for FPT. The true prevalence of FPT in this population of calves was 34.5 % (95 % BCI: 26.1–44.3) using a FPT cut-off point of 10 g IgG/L. This true prevalence increased to 66.3 (95 % BCI: 56.9–74.8) and 88.9 % (95 % BCI: 83.1–93.2) at FPT cut-off points of respectively 18 and 25 g IgG/L serum. The Bland-Altman plot comparing the RID and CE methods, revealed that the serum IgG concentrations obtained by RID were on average 2.25 (95 % CI 1.62–2.88) g/L higher than those measured by CE. Optimal cut-off values for CE, corresponding to the FPT values as measured by RID of 10, 18, and 25 g IgG/L serum, were respectively 10, 15, and 20 g IgG/L. The overall diagnostic accuracy of the three diagnostic tests was comparable at the FPT cut-off point of 10 g IgG/L serum (i.e. 85 %). At higher cut-off points for FPT, the RID and CE assays presumably performed better that the Bx method. In conclusion, we demonstrated that: (1) the CE method is a good alternative for the RID assay, the latter having important constraints when considering its practicality, and (2) the Bx method is a cheap and user-friendly indirect method to evaluate the FPT in new-born Belgian Blue beef calves.

Robust humoral immune response against rabies virus in rabbits and guinea pigs immunized with plasmid DNA vectors encoding rabies virus glycoproteins – An approach to the production of polyclonal antibody reagents

DNA-based immunization has been previously shown to be an efficient approach to induce robust immunity against infectious diseases in animals and humans. The advantages of DNA vaccines are simplicity of their construction and production, low cost, high stability, and ability to elicit a full spectrum of immune responses to target antigens. The goals of this study were (i) to assess the antibody immune response to rabies virus glycoproteins (rGPs) in rabbits and guinea pigs after intramuscular immunization with pTargeT and pVAC2-mcs mammalian expression vectors encoding either the wild-type (WT) or codon-optimized (cOPT) rGP genes; and (ii) to prepare in-house rabbit anti-rGP polyclonal antibody reagents suitable for in Single Radial Immunodiffusion (SRID) and Indirect Fluorescent Antibody (IFA) assays. The maximum antibody responses against rabies virus in rabbits and guinea pigs were observed after immunization series with 500 μg/dose of pVAC2-mcs vector encoding either the WT or cOPT rGP genes adjuvanted with Emulsigen-D. No significant difference in the anti-rabies virus neutralizing antibody titers was observed in rabbits immunized with the WT and cOPT rGPs. The in-house rabbit anti-rGP polyclonal antibody reagents reacted comparable to the current reference reagents in SRID and IFA assays. The results of the study demonstrated that the DNA immunization of animals with the WT or cOPT rGPs is a promising approach to either induction of high anti-rabies virus neutralizing antibody titers in vivo or for production of polyclonal antibody reagents against rabies.

Diagnostic accuracy of digital Brix and serum total protein refractometers in estimating different passive immunity levels in dairy calves

The aim of this study was to determine the diagnostic accuracy of digital serum total protein (TP) and digital Brix refractometers in estimating different passive immunity levels (<10, <18, <25 mg/mL) in dairy calves. Blood samples were collected from 260 apparently healthy Holstein calves, aged 2–7 days. Serum IgG concentrations were measured using digital Brix and TP refractometers and the radial immunodiffusion (RID) assay, as the gold standard. Data were analyzed by a receiver operating characteristics (ROC) analysis, the area under the ROC curves (AUC) and Cohen's kappa (κ). Optimal thresholds were determined as < 8.4, < 9.0 and < 9.4% for the digital Brix refractometer, and < 5.0, < 5.4 and < 5.8 g/dL for the serum TP refractometer in estimating IgG concentrations of < 10, < 18, < 25 mg/mL, respectively. The sensitivity (Se) and specificity (Sp) of the Brix refractometer were 96.3% and 88.8% for < 8.4% Brix, 97.0% and 83.4% for < 9.0% Brix, and 85.5% and 77.8% for < 9.4% Brix, respectively. The Se and Sp of the serum TP refractometer were 96.3% and 90.1% for < 5.0 g/dL, 91.0% and 89.6% for < 5.4 g/dL, 79.6% and 85.2% for < 5.8 g/dL, respectively. The discriminant ability of the refractometers was moderately accurate in estimating IgG concentrations of < 10 and < 18 mg/mL, and highly accurate in estimating IgG concentrations of < 25 mg/mL. Both refractometers substantially agreed with RID-IgG results and almost perfectly agreed with each other. In conclusion, the digital Brix and digital serum TP refractometers offer a good utility for determining different passive immunity levels in dairy calves.

Prevalence of antibodies in Iraqi Urinary Tract Infection patients using radial immunodiffusion (RID) assay

Urinary Tract Infection (UTI) is an infection every place in the urinary tract that may be in the urethra, bladder, or kidneys by microbes. Greatest UTIs are affected by bacteria, but some are affected by fungi and, in rare cases, by viruses. UTI is the most significant common infection in humans. This study deals with the prevalence of antibodies in UTI patients; this study aims to determine the level of antibodies in UTI patients and compare with healthy controls by using the radial immunodiffusion (RID) test. The study was done during the period November 2019 to April 2020 on UTI Iraqi patients. The study included 40 patients and 20 healthy controls. Results show UTI infection occurs in females more than males also; the mean age is 40 years. All the patients with UTI showed decreased IgM serum levels and increased IgG compared with the control group. IgG, IgG, and IgM showed high significance between two UTI patients and the control group groups, while IgM doesn’t show significant differences between study groups.

Suitability of Protein Content Measured by MilkoScan FT-Plus Milk Analyzer to Evaluate Bovine and Ovine Colostrum Quality.

Simple SummaryA rapid and reliable method for assessing colostrum quality is needed to ensure enough immunoglobulins available to offspring in order to avoid the failure of the passive transfer of immunity. This study evaluates MilkoScan FT-plus as a rapid tool for the evaluation of immunoglobulin content indirectly by protein content measurement and thus colostrum quality with a particular focus on bovine and ovine colostrum. The results indicate that MilkoScan FT-plus has good agreement with the reference method for the measurement of immunoglobulin concentration in colostrum represented by the radial immunodiffusion method.The objective of this study was to evaluate MilkoScan FT-plus for the estimation of the immunoglobulin G (IgG) content in bovine and ovine colostrum. Between April and May 2016, a total of 94 colostrum samples (54 from Simmental dairy cows and 39 from Sarda ewes) were collected within 6 h (T0) and after 24 h (T24) from parturition. Colostrum samples were subjected to the radial immunodiffusion (RID) assay for the quantification of IgG and to MilkoScan FT-plus for the estimation of protein content (TP, %), which was then used as an indirect method for the evaluation of colostrum quality. To compare the two methods, correlation and regression analysis of IgG quantification by RID and protein (%) content estimation by MilkoScan FT-plus data was performed using Procedure CORR and Procedure REG of SAS, respectively (version 9.3, SAS Institute Inc., Cary, NC, USA). Thresholds for the classification of good colostrum quality (as determined by RID assay, the gold standard method) were set at 50 g of IgG/L in cows and 20 g of IgG/L in ewes. The concentration of IgG in bovine colostrum assayed by RID showed a variation ranging from 41.45 to 199.97 g/L with an average of 99.85 ± 40.84 g/L at T0, and from 2.83 to 75.93 g/L with an average of 19.76 ± 19.01 g/L at T24. Regarding ovine colostrum, the concentration of IgG assayed by RID ranged from 34.45 to 156.32 g/L with an average value of 77.82 ± 37.58 g/L at T0, and from 5.6 to 69.74 g/L with an average of 27.90 ± 19.81 g/L at T24. Colostrum TP ranged from 3.70 to 23.96% for bovine colostrum and 6.32 to 22.88% for ovine colostrum using MilkoScan FT-plus. MilkoScan FT-plus and RID data were highly and significantly correlated (r = 0.91 for bovine and r = 0.94 for ovine colostrum), and regression analysis showed a strong relationship between IgG concentration provided by RID assay and TP provided by MilkoScan FT-plus (R2 = 0.84 and 0.88 for bovine and ovine, respectively). Optimal cut-off points for the greatest accuracy of TP (%) determined by MilkoScan FT-plus were 12.8% in cows [with 88.9% sensitivity (Se) and 100% specificity (Sp)] and 9% in ewes (with 96.7% Se and 100% Sp). In conclusion, these outcomes indicate that MilkoScan FT-plus as an indirect method may be a reliable tool for the estimation of the total IgG concentration and quality in bovine and ovine colostrum. Moreover, the cut-off levels of 12.8% for bovine and 9% for ovine of TP, seem sufficient to ensure that all poor-quality colostrum can be classified as such, with only a low proportion of good-quality colostrum being misclassified as poor-colostrum, thereby increasing the probability of delivering good-quality colostrum to new-born calves and lambs.