Sharif S. Aly

Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves.

BackgroundBovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated.ResultsThe four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined.ConclusionsFour analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

Development of a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves

Several clinical scoring systems for diagnosis of bovine respiratory disease (BRD) in calves have been proposed. However, such systems were based on subjective judgment, rather than statistical methods, to weight scores. Data from a pair-matched case-control study on a California calf raising facility was used to develop three novel scoring systems to diagnose BRD in preweaned dairy calves. Disease status was assigned using both clinical signs and diagnostic test results for BRD-associated pathogens. Regression coefficients were used to weight score values. The systems presented use nasal and ocular discharge, rectal temperature, ear and head carriage, coughing, and respiratory quality as predictors. The systems developed in this research utilize fewer severity categories of clinical signs, require less calf handling, and had excellent agreement (Kappa > 0.8) when compared to an earlier scoring system. The first scoring system dichotomized all clinical predictors but required inducing a cough. The second scoring system removed induced cough as a clinical abnormality but required distinguishing between three levels of nasal discharge severity. The third system removed induced cough and forced a dichotomized variable for nasal discharge. The first system presented in this study used the following predictors and assigned values: coughing (induced or spontaneous coughing, 2 points), nasal discharge (any discharge, 3 points), ocular discharge (any discharge, 2 points), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C or 102.5°F, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized “BRD positive” if their total score was ≥4. This system correctly classified 95.4% cases and 88.6% controls. The second presented system categorized the predictors and assigned weights as follows: coughing (spontaneous only, 2 points), mild nasal discharge (unilateral, serous, or watery discharge, 3 points), moderate to severe nasal discharge (bilateral, cloudy, mucoid, mucopurlent, or copious discharge, 5 points), ocular discharge (any discharge, 1 point), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized “BRD positive” if their total score was ≥4. This system correctly classified 89.3% cases and 92.8% controls. The third presented system used the following predictors and scores: coughing (spontaneous only, 2 points), nasal discharge (any, 4 points), ocular discharge (any, 2 points), ear and head carriage (ear droop or head tilt, 5 points), fever (≥39.2°C, 2 points), and respiratory quality (abnormal respiration, 2 points). Calves were categorized “BRD positive” if their total score was ≥5. This system correctly classified 89.4% cases and 90.8% controls. Each of the proposed systems offer few levels of clinical signs and data-based weights for on-farm diagnosis of BRD in dairy calves.

Correlation between Herrold egg yolk medium culture and real-time quantitative polymerase chain reaction results for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental samples

Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearmans rank correlation between Ct and CFU was good (–0.66) in fresh and thawed pooled fecal samples, and excellent (–0.76) and good (–0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (–0.53) to excellent (–0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

Randomized noninferiority clinical trial evaluating 3 commercial dry cow mastitis preparations: I. Quarter-level outcomes

The study objective was to compare the efficacy of 3 commercial dry cow mastitis formulations regarding quarter-level prevalence of intramammary infections (IMI) postcalving, cure of preexisting infections over the dry period, prevention of new infections during the dry period, and risk for a clinical mastitis case between calving and 100d in milk (DIM). A total of 1,091 cows (4,364 quarters) from 6 commercial dairy herds in 4 different states (California, Iowa, Minnesota, and Wisconsin) were enrolled and randomized to 1 of the 3 treatments at dry-off: Quartermaster (QT; 1,000,000 IU of procaine penicillin G and 1 g of dihydrostreptomycin; Pfizer Animal Health, New York, NY), Spectramast DC (SP; 500 mg of ceftiofur hydrochloride; Pfizer Animal Health), or ToMorrow Dry Cow (TM; 300mg of cephapirin benzathine; Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO). Quarter milk samples were collected for routine bacteriological culture before dry cow therapy treatment at dry-off, 0 to 6 DIM, and 7 to 13 DIM and an on-farm record-keeping system was used to retrieve data on clinical mastitis cases. Noninferiority analysis was used to evaluate the effect of treatment on the primary outcome, risk for a bacteriological cure during the dry period. Multivariable logistic regression techniques were used to describe the effect of treatment on risk for presence of IMI postcalving and risk of a new IMI during the dry period. Cox proportional hazards regression was used to describe the effect of treatment on the risk and time for quarters to experience an episode of clinical mastitis between calving and 100 DIM. The overall crude quarter-level prevalence of infection at dry-off was 19.2%. The most common pathogen isolated from milk samples at dry-off was coagulase-negative Staphylococcus, followed by Aerococcus spp. and other Streptococcus spp. Noninferiority analysis showed no effect of treatment on risk for a cure between dry-off and calving [least squares means (LSM): QT=93.3%, SP=92.6%, and TM=94.0%] and secondary analysis showed no effect of treatment on risk for presence of an IMI at 0 to 6 DIM (LSM: QT=16.5%, SP=14.1%, and TM=16.0%), risk for development of a new IMI between dry-off and 0 to 6 DIM (LSM: QT=14.8%, SP=12.3%, and TM=14.2%), or risk of experiencing a clinical mastitis event between calving and 100 DIM (LSM: QT=5.3%, SP=3.8%, and TM=4.1%). In conclusion, no difference was observed in efficacy among the 3 products evaluated when assessing the aforementioned quarter-level outcomes.

Association between Mycobacterium avium subspecies paratuberculosis infection and milk production in two California dairies

The association between Mycobacterium avium ssp. paratuberculosis (MAP) and milk production was estimated on 2 California dairies using longitudinal data from 5,926 cows. Both study herds had moderate MAP seroprevalence, housed cows in freestalls, and had Johnes disease control programs. Cow MAP status was determined using both serum ELISA and fecal culture results from cows tested at dry-off and from whole-herd tests. Potential confounders were evaluated based on a causal diagram. Mixed models with 2 functions (splines) for days in milk (DIM) representing milk production pre- and postpeak used in similar studies were further modified to use each cows observed DIM at peak and lactation length. Cows that were seropositive produced 2.5kg less 4% fat-corrected milk (FCM) per day than their seronegative herdmates. In addition, cows that were fecal-culture positive by liquid culture and confirmed by PCR produced 2.2kg less 4% FCM per day than their fecal-culture negative herdmates. The decrease in milk production in MAP test-positive compared with test-negative cows started in the second lactation. A switch in MAP status in either ELISA or fecal culture results from positive to negative had no significant association with milk production. Modified DIM functions that used the observed DIM at peak had better model fit than another function that assumed a fixed peak at 60 DIM. Cows that tested positive for MAP on serum ELISA or fecal culture produced less milk than cows that tested negative, and the association between MAP and milk production was not confounded by mastitis, elevated somatic cell counts, or uterine or metabolic cow conditions.

Blood calcium dynamics after prophylactic treatment of subclinical hypocalcemia with oral or intravenous calcium

Total serum Ca dynamics and urine pH levels were evaluated after prophylactic treatment of subclinical hypocalcemia after parturition in 33 multiparous Jersey × Holstein crossbreed cows. Cows were blocked according to their calcemic status at the time of treatment [normocalcemic (8.0-9.9 mg/dL; n = 15) or hypocalcemic (5.0-7.9 mg/dL; n = 18)] and randomly assigned to 1 of 3 treatments: control [no Ca supplementation (n = 11)]; intravenous Ca [Ca-IV (n = 11), 500 mL of 23% calcium gluconate (10.7 g of Ca and 17.5 g of boric acid as a solubilizing agent; Durvet, Blue Springs, MO)]; or oral Ca [Ca-Oral (n = 11), 1 oral bolus (Bovikalc bolus, Boehringer Ingelheim, St. Joseph, MO) containing CaCl2 and CaSO4 (43 g of Ca) 2 times 12h apart]. Total serum Ca levels were evaluated at 0, 1, 2, 4, 8, 12, 16, 20, 24, 36, and 48 h, and urine pH was evaluated at 0, 1, 12, 24, 36, and 4 8h after treatment initiation. Total serum Ca levels were higher for Ca-IV than for control and Ca-Oral cows at 1, 2, and 4h after treatment initiation, but lower than Ca-Oral cows at 20, 24, and 36 h and lower than control cows at 36 and 48 h. At 1h after treatment initiation, when serum Ca levels for Ca-IV cows peaked (11.4 mg/dL), a greater proportion of Ca-IV (n = 8) cows had total serum Ca levels >10mg/dL than control (n = 0) and Ca-Oral (n = 1) cows. At 24h after treatment initiation, when Ca-IV cows reached the total serum Ca nadir (6.4 mg/dL), a greater proportion of Ca-IV (n = 10) cows had serum Ca levels <8 mg/dL than control (n = 5) and Ca-Oral (n = 2) cows. Treatment, time, and treatment × time interaction were significant for urine pH. Mean urine pH was lower for Ca-Oral cows (6.69) than for control (7.52) and Ca-IV (7.19) cows. Urine pH levels at 1h after treatment were lower for Ca-IV cows compared with both control and Ca-Oral cows, a finding likely associated with the iatrogenic administration of boric acid added as a solubilizing agent of the intravenous Ca solution used. At 12, 24, and 36 h, urine pH levels were lower for Ca-Oral cows compared with both control and Ca-IV cows. This was expected because the oral Ca supplementation used (Bovikalc) is designed as an acidifying agent. Wide fluctuations in blood Ca were observed after prophylactic intravenous Ca supplementation. The implications for milk production and animal health, if any, of these transient changes in total serum Ca have yet to be evaluated.

Reliability of environmental sampling to quantify Mycobacterium avium subspecies paratuberculosis on California free-stall dairies.

The reliability of environmental sampling to quantify Mycobacterium avium ssp. paratuberculosis (MAP) based on collector and time was evaluated. Fecal slurry samples were collected using a standardized protocol simultaneously by 2 collectors of different experience levels. Samples were collected from 30 cow pens on 4 dairies every other day on 3 occasions while cow movements between pens were minimal. The 4 study herds had moderate MAP seroprevalence and were housed in free-stall dairies in central California. Results of testing the environmental samples for MAP using PCR and culture were strongly correlated. The reliability of environmental sampling simultaneously by different collectors as estimated by the intraclass correlation coefficient was excellent (81%) for PCR and good (67%) for culture and may justify comparison of quantitative results of samples collected by different investigators. The reliability of environmental sampling over a 5-d period was good (67 and 64% for PCR and culture results, respectively), which justifies the utility of environmental sampling to identify pens with a high MAP bioburden between routine cow pen changes on a dairy. Environmental sampling of free-stall pens using the standardized sampling protocol yielded comparable PCR and culture results across collectors with different experience levels and at different times within a 5-d period.

Synergistic Effects of Lactic Acid and Sodium Dodecyl Sulfate to Decontaminate Escherichia coli O157:H7 on Cattle Hide Sections

The objective of this study was to investigate the antibacterial properties of chitosan acetate (CA), sodium dodecyl sulfate (SDS), lactic acid (LA) and their synergism when combined against a nontoxigenic strain of Escherichia coli O157:H7. Treatments that significantly reduced the concentration of E. coli O157:H7 in vitro by more than two logs were further investigated using a cattle hide decontamination model. In vitro treatments included CA (1% chitosan in 1% acetic acid vol/vol), SDS (1% vol/vol), SDS (2% vol/vol), LA (1% vol/vol), CA-SDS combination (1% chitosan in 1% acetic acid vol/vol mixed with 1% SDS vol/vol), and LA-SDS combination in two different concentrations (1% LA mixed with 1% SDS vol/vol, and 1% LA mixed with 2% SDS vol/vol). Butterfields Phosphate Buffer water was used as a control. The antibacterial effect of 1% CA solution alone and in combination with 1% SDS in vitro resulted in a 1.8 and 1.7 log colony-forming units (CFU)/mL reduction, respectively (p<0.05). Only 1% LA, 1% SDS, 2% SDS and their combinations resulted in a >2 log reduction in E. coli O157:H7. On hide sections, both 1% LA-1% SDS and 1% LA-2% SDS combinations significantly (p<0.05) reduced E. coli O157:H7 concentration by 4.6 and 4.7 log CFU/ cm(2) greater than the control, respectively. There was no significant difference in the antibacterial effect of 1% LA compared to the control, 2% SDS compared to the control, or 1% LA compared to 2% SDS. Hence, the antibacterial efficacy of 1% LA against E. coli O157:H7 on hide sections was significantly enhanced when combined with 1% SDS. Results of this study support the use of low concentration LA-SDS combination as a hide wash to reduce the risk of E. coli O157:H7 contamination.

Prevalence and level of enterohemorrhagic escherichia coli in culled dairy cows at harvest

The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n = 300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm(2) in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (κ = 0.58, P < 0.001), EHEC O121 (κ = 0.50, P < 0.001), and EHEC O157 (κ = 0.40, P < 0.001). No significant agreement was observed between NS and Atlas results or between culture and Atlas results. Detection of an EHEC serogroup in fecal samples was significantly associated with detection of the same EHEC serogroup in hide samples for EHEC O26 (P = 0.001), EHEC O111 (P = 0.002), EHEC O121 (P < 0.001), and EHEC-6 (P = 0.029) based on NS detection and for EHEC O121 (P < 0.001) based on detection by culture. This study provides evidence that non-O157 EHEC are ubiquitous on hides of culled dairy cattle and that feces are an important source of non-O157 EHEC hide contamination.

Sensitivity and specificity of on-farm scoring systems and nasal culture to detect bovine respiratory disease complex in preweaned dairy calves

The California (CA) and Wisconsin (WI) clinical scoring systems have been proposed for bovine respiratory disease complex (BRDC) detection in preweaned dairy calves. The screening sensitivity (SSe), for estimating BRDC prevalence in a cohort of calves, diagnostic sensitivity (DSe), for confirming BRDC in ill calves, and specificity (Sp) were estimated for each of the scoring systems, as well as for nasal swab cultures for aerobic bacteria and mycoplasma species. Thoracic ultrasound and auscultation were used as the reference standard tests interpreted in parallel. A total of 536 calves (221 with BRDC and 315 healthy) were sampled from 5 premises in California. The SSe of 46.8%, DSe of 72.6%, and Sp of 87.4% was determined for the CA system. The SSe of 46.0%, DSe of 71.1%, and Sp of 91.2% was determined for the WI system. For aerobic culture, the SSe was 43.4%, DSe was 52.6%, and Sp was 71.3%; for Mycoplasma spp. culture, the SSe was 57.5%, DSe was 68.9%, and Sp was 59.7%. The screening and diagnostic sensitivities of the scoring systems were not significantly different but the Sp of the WI system was greater by 3.8%. Scoring systems can serve as rapid on-farm tools to determine the burden of BRDC in preweaned dairy calves. However, users may expect the SSe to be less than the DSe when confirming BRDC in an ill calf.