Ibrahim Elsohaby

Evaluation of Digital and Optical Refractometers for Assessing Failure of Transfer of Passive Immunity in Dairy Calves

Background Failure of transfer of passive immunity (FTPI) is the underlying predisposing risk factor for most early losses in dairy calves. Refractometers, either optical or digital, can be used to assess FTPI as a part of calf health monitoring program on dairy operations. Objectives To evaluate the performance of and differences between digital Brix and optical refractometers for assessing FTPI in dairy calves. Animals Two hundred Holstein calves from 1 to 11 days of age. Methods A cross‐sectional study was designed to measure serum IgG concentration by radial immunodiffusion (RID) assay, digital Brix and optical refractometers. The correlation coefficients (r) between the 2 refractometers were plotted against each other and against the measured IgG concentration from RID. The Se, Sp, and accuracy of digital Brix and optical refractometers for assessing FTPI using previously recommended cut‐offs were calculated. A receiver operating characteristic curve was created and used to identify the optimal cut‐off for this dataset. Results The RID IgG concentration was positively correlated with digital Brix (r = 0.79) and optical (r = 0.74) refractometers. The best combination of Se (85.5%), Sp (82.8%), and accuracy (83.5%) of digital Brix refractometer was at 8.3%Brix. For optical refractometer, the best combination of Se (80%), Sp (80.7%), and accuracy (80.5%) was at 5.5 g/dL. Conclusions and Clinical Importance Both refractometers exhibited utility in assessing FTPI in dairy calves.

Measurement of serum immunoglobulin G in dairy cattle using Fourier-transform infrared spectroscopy: A reagent free approach

Simple, rapid and cost-effective methods are sought for measuring immunoglobulin G (IgG) concentrations in bovine serum, which can be applied for diagnosis of failure of transfer of passive immunity (FTPI). The aim of the present study was to investigate the potential use of Fourier-transform infrared (FTIR) spectroscopy, with partial least squares (PLS) regression, to measure IgG concentrations in bovine serum. Serum samples collected from calves and adult cows were tested in parallel by radial immunodiffusion (RID) assay and FTIR spectroscopy. The sample IgG concentrations obtained by the RID method were linked to pre-processed spectra and divided into two sets: a combined set and a test set. The combined set was used for building a calibration model, while the test set was used to assess the predictive ability of the calibration model, resulting in a root mean squared error of prediction (RMSEP) of 307.5 mg/dL. The concordance correlations between the IgG measured by RID and predicted by FTIR spectroscopy were 0.96 and 0.93 for the combined and test data sets, respectively. Analysis of the data using the Bland-Altman method did not show any evidence of systematic bias between FTIR spectroscopy and RID methods for measurement of IgG. The clinical applicability of FTIR spectroscopy for diagnosis of FTPI was evaluated using the entire data set and showed a sensitivity of 0.91 and specificity of 0.96, using RID as the reference standard. The FTIR spectroscopy method, described in the present study, demonstrates potential as a rapid and reagent-free tool for quantification of IgG in bovine serum, as an aid to diagnosis of FTPI in calves.

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.

Quantification of bovine immunoglobulin G using transmission and attenuated total reflectance infrared spectroscopy

In this study, we evaluated and compared the performance of transmission and attenuated total reflectance (ATR) infrared (IR) spectroscopic methods (in combination with quantification algorithms previously developed using partial least squares regression) for the rapid measurement of bovine serum immunoglobulin G (IgG) concentration, and detection of failure of transfer of passive immunity (FTPI) in dairy calves. Serum samples (n = 200) were collected from Holstein calves 1–11 days of age. Serum IgG concentrations were measured by the reference method of radial immunodiffusion (RID) assay, transmission IR (TIR) and ATR-IR spectroscopy-based assays. The mean IgG concentration measured by RID was 17.22 g/L (SD ±9.60). The mean IgG concentrations predicted by TIR and ATR-IR spectroscopy methods were 15.60 g/L (SD ±8.15) and 15.94 g/L (SD ±8.66), respectively. RID IgG concentrations were positively correlated with IgG levels predicted by TIR (r = 0.94) and ATR-IR (r = 0.92). The correlation between 2 IR spectroscopic methods was 0.94. Using an IgG concentration <10 g/L as the cut-point for FTPI cases, the overall agreement between TIR and ATR-IR methods was 94%, with a corresponding kappa value of 0.84. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for identifying FTPI by TIR were 0.87, 0.97, 0.91, 0.95, and 0.94, respectively. Corresponding values for ATR-IR were 0.87, 0.95, 0.86, 0.95, and 0.93, respectively. Both TIR and ATR-IR spectroscopic approaches can be used for rapid quantification of IgG level in neonatal bovine serum and for diagnosis of FTPI in dairy calves.

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.

Listeria monocytogenes in raw milk, milking equipment and dairy workers: Molecular characterization and antimicrobial resistance patterns

OBJECTIVES The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst L. monocytogenes isolated from raw milk, milking equipment, and hand swabs from workers in dairy farms. METHODS A total of 300 samples of raw milk, milking equipment, and hand swabs were collected from four dairy farms to examine the presence of Listeria species. Suspected isolates were further identified by VITEK-2 system and Polymerase Chain Reaction (PCR). Antimicrobial susceptibility of the L. monocytogenes isolates was determined, and genotyping analysis was performed by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). RESULTS Listeria spp. was isolated from 79 (26.3%) of the 300 samples, including 29 (36.7%), 32 (40.5%), and 18 (22.8%) isolates found in raw milk, milking equipment, and hand swabs, respectively. L. monocytogenes was the most common isolated (87.3%) species, while the remaining Listeria isolates were L. innocua (12.7%). Among the 69 L. monocytogenes isolates, 42 (60.8%) showed the mutual presence of hlyA, prfA, inlA, and inlB virulence-associated genes. L. monocytogenes isolates from raw milk, milking equipment, and hand swabs showed high genetic relatedness. The potentially virulent L. monocytogenes isolates were most frequently resistance to tetracycline and clindamycin (81%, each) followed by rifampicin (71.4%), whereas, antimicrobial susceptibility was most frequently observed for ampicillin, levofloxacin, moxifloxacin, linezolid, and tigecycline (100%, each). Furthermore, 88% of L. monocytogenes isolates showed multidrug-resistance. CONCLUSIONS The findings of this study show that the contamination of dairy farms with L. monocytogenes is relatively high, and highlight the emergence of multi-drug resistant L. monocytogenes in dairy farms. However, ampicillin is a good choice for treatment of listeriosis in the study area.

Application of transmission infrared spectroscopy and partial least squares regression to predict immunoglobulin G concentration in dairy and beef cow colostrum

The objective of this study was to explore the potential of transmission infrared (TIR) spectroscopy in combination with partial least squares regression (PLSR) for quantification of dairy and beef cow colostral immunoglobulin G (IgG) concentration and assessment of colostrum quality. A total of 430 colostrum samples were collected from dairy (n = 235) and beef (n = 195) cows and tested by a radial immunodiffusion (RID) assay and TIR spectroscopy. Colostral IgG concentrations obtained by the RID assay were linked to the preprocessed spectra and divided into combined and prediction data sets. Three PLSR calibration models were built: one for the dairy cow colostrum only, the second for beef cow colostrum only, and the third for the merged dairy and beef cow colostrum. The predictive performance of each model was evaluated separately using the independent prediction data set. The Pearson correlation coefficients between IgG concentrations as determined by the TIR-based assay and the RID assay were 0.84 for dairy cow colostrum, 0.88 for beef cow colostrum, and 0.92 for the merged set of dairy and beef cow colostrum. The average of the differences between colostral IgG concentrations obtained by the RID- and TIR-based assays were -3.5, 2.7, and 1.4 g/L for dairy, beef, and merged colostrum samples, respectively. Further, the average relative error of the colostral IgG predicted by the TIR spectroscopy from the RID assay was 5% for dairy cow, 1.2% for beef cow, and 0.8% for the merged data set. The average intra-assay CV% of the IgG concentration predicted by the TIR-based method were 3.2%, 2.5%, and 6.9% for dairy cow, beef cow, and merged data set, respectively.The utility of TIR method for assessment of colostrum quality was evaluated using the entire data set and showed that TIR spectroscopy accurately identified the quality status of 91% of dairy cow colostrum, 95% of beef cow colostrum, and 89% and 93% of the merged dairy and beef cow colostrum samples, respectively. The results showed that TIR spectroscopy demonstrates potential as a simple, rapid, and cost-efficient method for use as an estimate of IgG concentration in dairy and beef cow colostrum samples and assessment of colostrum quality. The results also showed that merging the dairy and beef cow colostrum sample data sets improved the predictive ability of the TIR spectroscopy.

Application of laboratory and portable attenuated total reflectance infrared spectroscopic approaches for rapid quantification of alpaca serum immunoglobulin G

The objective of this study was to develop and compare the performance of laboratory grade and portable attenuated total reflectance infrared (ATR-IR) spectroscopic approaches in combination with partial least squares regression (PLSR) for the rapid quantification of alpaca serum IgG concentration, and the identification of low IgG (<1000 mg/dL), which is consistent with the diagnosis of failure of transfer of passive immunity (FTPI) in neonates. Serum samples (n = 175) collected from privately owned, healthy alpacas were tested by the reference method of radial immunodiffusion (RID) assay, and laboratory grade and portable ATR-IR spectrometers. Various pre-processing strategies were applied to the ATR-IR spectra that were linked to corresponding RID-IgG concentrations, and then randomly split into two sets: calibration (training) and test sets. PLSR was applied to the calibration set and calibration models were developed, and the test set was used to assess the accuracy of the analytical method. For the test set, the Pearson correlation coefficients between the IgG measured by RID and predicted by both laboratory grade and portable ATR-IR spectrometers was 0.91. The average differences between reference serum IgG concentrations and the two IR-based methods were 120.5 mg/dL and 71 mg/dL for the laboratory and portable ATR-IR-based assays, respectively. Adopting an IgG concentration <1000 mg/dL as the cut-point for FTPI cases, the sensitivity, specificity, and accuracy for identifying serum samples below this cut point by laboratory ATR-IR assay were 86, 100 and 98%, respectively (within the entire data set). Corresponding values for the portable ATR-IR assay were 95, 99 and 99%, respectively. These results suggest that the two different ATR-IR assays performed similarly for rapid qualitative evaluation of alpaca serum IgG and for diagnosis of IgG <1000 mg/dL, the portable ATR-IR spectrometer performed slightly better, and provides more flexibility for potential application in the field.

Molecular identification of avian influenza virus subtypes H5N1 and H9N2 in birds from farms and live bird markets and in respiratory patients

Background Avian influenza viruses (AIVs) have been endemic in Egypt since 2006, and the co-circulation of high-pathogenic avian influenza H5N1 and low-pathogenic avian influenza H9N2 subtypes in poultry has been reported; therefore, Egypt is considered a hotspot for the generation of new subtypes and genotypes. We aimed to characterize AIVs circulating on commercial farms and in live bird markets (LBMs) during the winters of 2015 and 2016 in the study area and to identify H5N1 and H9N2 viruses in respiratory patients. Methods In total, 159 samples were collected from ducks, pigeons and quails on farms (n = 59) and in LBMs (n = 100) and screened by real-time RT-PCR for H5N1 and H9N2 subtypes. Clinical and postmortem examination was carried out on birds from the farms. Positive H5N1 samples were sequenced and analysed for mutations. Tracheal swabs were also collected from 89 respiratory patients admitted to respiratory hospitals in the same study area. Results Overall, H5N1 was identified in 13.6% of birds from farms, while it was detected in 17% of birds in LBMs. Subtype H9N2 was only identified from pigeons on farms (6.5%) and LBMs (11.4%). Sequencing of the haemagglutination gene (HA) in nine representative H5N1 isolates revealed a multi-basic amino acid motif at the cleavage site (321-PQGEKRRKKR/GLF-333), which is characteristic of highly pathogenic AIV, in five of our isolates, while the other four isolates showed an amino acid substitution (Q322K) at this cleavage site to make it (321-P K GEKRRKKR/GLF-333). All the isolates belonged to clade 2.2.1.2, and a comparison of HA sequences at the amino acid level showed 98.8–100% homology among the nine isolates, while they showed 94.1–96.1% identity with reference strains and the commonly used vaccine strain in Egypt. Out of 89 respiratory patients, 3.4% were positive for H5N1 and no patients were positive for H9N2. Discussion Our results indicated the circulation of the endemic H5N1 and H9N2 viruses among poultry in 2015 and 2016. Birds on farms and in LBMs are reservoirs playing a role in the dissemination of the virus and producing a public health risk. The application of proper hygienic measures in farms and LBMs to control the exposure of birds and humans to the source of infection along with continuous monitoring of the circulating viruses will provide information on understanding the evolution of the viruses for vaccine studies.

Effect of heat-treatment on accuracy of infrared spectroscopy and digital and optical Brix refractometers for measuring immunoglobulin G concentration in bovine colostrum

Background Heat‐treatment of colostrum is a method developed to reduce calf exposure to pathogens. Infrared (IR) spectroscopy and Brix refractometers can be used for measuring colostral IgG concentration and assessing colostrum quality. Objectives To determine the impact of heat‐treatment on accuracy of IR spectroscopy and Brix refractometers for measuring colostral IgG concentration and assessing colostrum quality before and after heat‐treatment. Animals A total of 60 Holstein dairy cows on 8 commercial dairy farms. Methods A cross‐sectional study was designed to determine the effect of heat‐treatment at 60°C and 63°C each for 30 and 60 minutes duration on colostral IgG concentration measured by the reference radial immunodiffusion (RID) assay, IR spectroscopy, and digital and optical refractometers. Results Colostrum IgG concentration significantly decreased after heat‐treatment at 63°C for 30 or 60 minutes as measured by RID, but the IgG values remained unchanged when measured by IR spectroscopy and refractometers. The lowest correlation coefficient found between IR spectroscopy (r = 0.70) and RID results was in colostrum heat‐treated at 63°C for 60 minutes. For digital (r = 0.48) and optical (r = 0.50) refractometers, the lowest correlation coefficient was at 63°C for 30 minutes when compared to RID. The accuracy of the IR spectroscopy, digital and optical Brix refractometers was decreased from 91.7 to 80%, 81.7 to 45%, and 80 to 45%, respectively, when colostrum heat‐treated at 63°C for 60 minutes. Conclusions and Clinical Importance Radial immunodiffusion, IR spectroscopy, and Brix refractometers exhibit utility for measuring IgG concentration when colostrum heat‐treated at 60°C but does not detect decrease IgG concentrations when heat‐treated at 63°C.