Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform.

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1ß and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1ß, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

Assessment of nebulisation of sodium ceftiofur in the treatment of calves naturally infected with bovine respiratory disease.

Twelve screened cases of bovine respiratory disease (BRD) in calves were enrolled. Six of the calves were treated intramuscularly with sodium ceftiofur (1 mg/kg), and six were treated with nebulised sodium ceftiofur (1 mg/kg). Comparative evaluation of the two therapeutic modalities was based on repetitive analysis of hematological profile of calves on days 0, 5, and 10 post-therapy. The mortality rate in the group of calves treated with the nebulised sodium ceftiofur was significantly (p < 0.001) lower, and their clinical and hematological parameters returned to normal significantly (p < 0.001) faster than in calves treated intramuscularly. Nebulisation of sodium ceftiofur is the most effective treatment in calves with BRD under field conditions. Nasal lavage fluid analysis indicating a high rise of neutrophil count and macrophages may be used as an alternative method to detect pulmonary inflammation in BRD-affected calves.

Genetic parameters estimated at receiving for circulating cortisol, immunoglobulin G, interleukin 8, and incidence of bovine respiratory disease in feedlot beef steers.

Bovine respiratory disease complex (i.e., shipping fever and bacterial bronchopneumonia) is a multifaceted respiratory illness influenced by numerous environmental factors and microorganisms. Bovine respiratory disease (BRD) is just one component of BRD complex. Because BRD is moderately heritable, it may be possible to reduce the incidence of BRD through genetic selection. The objectives of this study were to determine the heritability and associative genetic relationships among immune system traits (i.e., cortisol, total IgG, IgG isotypes, and IL-8) in cattle monitored for BRD incidence. At an average of 83 d after weaning (219 d age and mean = 221.7 kg [SD 4.34]), crossbred steer calves ( = 2,869) were received at a commercial feedlot in southeastern Colorado over a 2-yr period. At receiving, jugular blood samples were collected at 212 (yr 1) and 226 d (yr 2) of age for immune trait analyses. The BRD phenotype was defined as a binomial variable (0 = no and 1 = yes) and compared with immune system traits measured at receiving (prior to illness onset). An animal identified as BRD positive exhibited ≥ 2 clinical signs (i.e., eye or nasal discharge, cough, lethargy, rapid breathing, acute interstitial pneumonia, or acute upper respiratory syndrome and/or a rectal temperature > 39.7°C). Heritability and genetic correlation estimates for categorical variable BRD, cortisol, IgG, IgG1, IgG2, and IL-8 were estimated from a sire model using ASREML. Heritability estimates were low to moderate for BRD (0.17 ± 0.08), cortisol (0.13 ± 0.05), IgG (0.15 ± 0.05), IgG1 (0.11 ± 0.05), IgG2 (0.24 ± 0.06), and IL-8 (0.30 ± 0.06). A moderate negative genetic correlation was determined between BRD and cortisol ( = -0.19 ± 0.32). Moderate positive correlations were found between BRD with IgG (0.42 ± 0.28), IgG1 (0.36 ± 0.32), and IL-8 ( = 0.26 ± 0.26). Variation in the BRD phenotype and immune system traits suggested herd health improvement may be achieved through genetic selection.

Systematic Review of the Diagnostic Accuracy of Haptoglobin, Serum Amyloid A, and Fibrinogen versus Clinical Reference Standards for the Diagnosis of Bovine Respiratory Disease.

BACKGROUND: Bovine respiratory disease (BRD) is a worldwide animal health concern especially in feedlot, dairy, and veal calves. One of the greatest challenges is the absence of a gold standard for achieving an accurate antemortem diagnosis. Various blood markers, including the acute-phase proteins (AAP), have been proposed as potential valuable tools for BRD diagnosis. OBJECTIVES: To perform a systematic review of the literature to assess the accuracy of selected APP (haptoglobin [Hp], serum amyloid A [SAA], and fibrinogen [Fb]) as diagnostic tools for cattle with naturally occurring BRD when compared with clinical reference standards of diagnosis. METHODS: This review was performed with eligible studies selected from CAB Abstract and MEDLINE from 1946 to 2015, as well as the "gray literature." Methodological quality of included studies was assessed using the QUADAS-2 tool developed for diagnostic accuracy studies. The accuracy parameters sensitivity (Se) and specificity (Sp) were obtained from the articles or through contact with the authors when not directly reported. RESULTS: A total of 314 studies were identified, from them, 23 met inclusion criteria as diagnostic studies for naturally occurring BRD. Quality of studies showed high risk of bias for case selection (70% of articles) and unclear risk of bias for index test (65%), reference standard (74%), and flow and timing (61%). There were high concerns regarding applicability for case selection (61% of studies) and reference standards used for defining BRD (48%). The concerns regarding index test application were low (83% of the studies). Only 4-8 studies could be included in the meta-analysis for each APP. No pooled estimates or pooled accuracy measurements were performed due to the low number of studies and multiple differences between studies, including reference standard definitions. CONCLUSIONS AND CLINICAL IMPORTANCE: On the basis of these findings, it is not possible to make conclusions regarding the accuracy of APP for BRD diagnosis. The reporting of accuracy of APP for BRD detection is inconsistent among studies. Recommendations to improve capability for future meta-analyses in this area include reporting studies on diagnostic tests following the Standard for the Reporting of Diagnostic Accuracy Studies (STARD), as well as trying to standardize BRD definition across future studies.

A Systematic Review of Bovine Respiratory Disease Diagnosis Focused on Diagnostic Confirmation, Early Detection, and Prediction of Unfavorable Outcomes in Feedlot Cattle.

A large proportion of newly arrived feedlot cattle are affected with bovine respiratory disease (BRD). Economic losses could be reduced by accurate, early detection. This review evaluates the available literature regarding BRD confirmatory diagnostic tests, early detection methods, and modalities to estimate post-therapeutic prognosis or predict unfavorable or fatal outcomes. Scientific evidence promotes the use of haptoglobin to confirm BRD status. Feeding behavior, infrared thermography, and reticulorumen boluses are promising methods. Retrospective analyses of routinely collected treatment and cohort data can be used to identify cattle at risk of unfavorable outcome. Other methods have been reviewed but require further study.

Assessment of L-lactatemia as a predictor of respiratory disease recognition and severity in feedlot steers.

The bovine respiratory disease complex (BRD) is a major health issue in feedlot cattle and one of the primary reasons for antimicrobial use in the North American feedlot industry. The purpose of the present study was to assess blood L-lactate levels of feedlot steers at high risk of developing BRD during the early feeding period. Blood samples were obtained at initial processing and again after BRD confirmation (using bronchial lavage or thoracic ultrasound exam). The study involved 232 recently weaned steers received at a single research feedlot that were processed without metaphylactic antimicrobial treatment. Blood samples were obtained for determination of L-lactatemia and temperament scores (very quiet or stoic [score 1], average [score 2] and very excited [score 3]) were systematically assigned at initial processing. A subsample of calves that were later confirmed as cases of BRD were sampled at first pull (day 0), and at subsequent observation points on days 3, 6, 9 and 15 following initial BRD diagnosis for blood lactate determination as a potential indicator of subsequent death. The clinical BRD cumulative incidence in the cohort was 38% (87/232). Temperament was associated with the probability of becoming a BRD case during the early feeding period. Stoic or very excited calves showed 2.2 times higher odds (95%CI: 1.3, 3.8) of becoming BRD cases compared to calves with average temperament. The impact of L-lactatemia differed by temperament strata. In calves with a temperament score of 2 (average temperament) every 1-log unit increase of lactatemia at processing resulted in 1.9 times higher odds (95% CI: 1.2, 3.1) of becoming a BRD case; this relationship was not significant in calves with a score of either 1 or 3. Twenty-nine confirmed BRD cases were studied for the dynamic lactate assessment analysis. L-lactate at first pull was not significantly different between survivors (median 3.3mmol/L; range 0.8-7.8mmol/L) and non-survivors (median 2.7mmol/L; range: 1.6-5.4mmol/L) steers. However, the dynamic assessment of L-lactatemia was associated with the hazard of death using Cox proportional hazard survival analysis. A 1-log increase of lactatemia increased the hazard of dying prior to the next observation by a factor of 36.5 (95% CI: 3.5-381.6). For calves showing a normal temperament score (i.e. temperament score of 2), a misclassification cost term analysis was conducted to identify potential L-lactate test thresholds for identifying future BRD steers. When planned test usage was for informing decision of administering or not a metaphylactic treatment at processing, experts agreed that false-negative (not treating a calf that would have benefit from treatment) to false-positive (wrongfully treating a calf that would have remained healthy) health costs ratio ranged from 8:1 to 20:1. In this situation, a threshold of 5mmol/L would have best informed treatment decision. When using L-lactate for informing the type of antimicrobial used at processing, false-negative to false-positive health costs ratio ranging from 1:1 to 3:1 could be expected and, again, a L-lactate threshold of 5.0mmol/L would have minimized the costs associated with calves' misclassification and could be used to identify calves that would benefit from a more efficient metaphylactic treatment. This study provides an interesting perspective on the potential application of chute-side markers or diagnostic tests to stratify the risk of future pull for BRD in cattle during processing in order to adapt antimicrobial treatments accordingly.

Association of hematologic variables and castration status at the time of arrival at a research facility with the risk of bovine respiratory disease in beef calves.

OBJECTIVE: To determine the association of CBC variables and castration status at the time of arrival at a research facility with the risk of development of bovine respiratory disease (BRD). DESIGN: Retrospective cohort study. ANIMALS: 1,179 crossbred beef bull (n = 588) and steer (591) calves included in 4 experiments at 2 University of Arkansas research facilities. PROCEDURES: Calves underwent processing and treatments in accordance with the experiment in which they were enrolled. Castration status and values of CBC variables were determined at the time of arrival at the facilities. Calves were monitored to detect signs of BRD during a 42-day period. RESULTS: The areas under the receiving operator characteristic curves for CBC variables with significant contrast test results ranged from 0.51 (neutrophil count) to 0.67 (eosinophil count), indicating they were limited predictors of BRD in calves. The only CBC variables that had significant associations with BRD in calves as determined via multivariable logistic regression analysis were eosinophil and RBC counts. The odds of BRD for bulls were 3.32 times the odds of BRD for steers. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study indicated that low eosinophil and high RBC counts in blood samples may be useful for identification of calves with a high risk for development of BRD. Further research may be warranted to validate these variables for prediction of BRD in calves. Calves that were bulls at the time of arrival had a higher risk of BRD, versus calves that were steers at that time.

Effectiveness of sorting calves with high risk of developing bovine respiratory disease on the basis of serum haptoglobin concentration at the time of arrival at a feedlot.

OBJECTIVE: To evaluate serum haptoglobin concentration at feedlot arrival and subsequent performance and morbidity and mortality rates of calves that developed bovine respiratory disease. ANIMALS: 360 heifer calves and 416 steer and bull calves. PROCEDURES: Serum samples were obtained from cattle at the time of arrival to a feedlot (day -1) and analyzed for haptoglobin concentration. In experiment 1, calves were classified into groups with a low (< 1.0 µg/mL), medium (1.0 to 3.0 µg/mL), or high (> 3.0 µg/mL) serum haptoglobin concentration and allotted into pens on the basis of group. In experiment 2, calves were classified as having or not having detectable serum haptoglobin concentrations. RESULTS: In experiment 1, average daily gain from days 1 to 7 decreased as haptoglobin concentration increased. Dry-matter intake (DMI) from days 1 to 21 decreased with increasing haptoglobin concentration, and DMI typically decreased from days 1 to 63. Total bovine respiratory disease morbidity rate typically increased with increasing haptoglobin concentration. At harvest, no differences in carcass characteristics were observed on the basis of haptoglobin concentration. In experiment 2, cattle with measureable serum haptoglobin concentrations at arrival weighed less throughout the experiment, gained less from days 1 to 7, and had lower DMI from days 1 to 42. Overall morbidity rate was not different between groups, but cattle with detectable serum haptoglobin concentrations had higher odds of being treated 3 times. CONCLUSIONS AND CLINICAL RELEVANCE: Serum haptoglobin concentration in cattle at the time of feedlot arrival was not associated with overall performance but may have limited merit for making decisions regarding targeted prophylactic treatment.

Associations between the prevalence of Mollicutes and Mycoplasma bovis and health and performance in stocker calves.

A longitudinal cross-sectional time-series study was carried out to determine the prevalence of nasal mycoplasma carriage, serostatus and seroconversion, and to evaluate the associations between these parameters and health and performance in weaned beef calves during a 42-day feeding period. Nasal swabs and serum were collected on days 0 (arrival), 10, 42 and at the first incidence of bovine respiratory disease complex. The samples were evaluated for Mollicutes (by culture), Mycoplasma bovis (by PCR) and serum antibody to M. bovis. On day 0, 90.4 per cent of the calves were Mollicutes nasal culture-positive. The seroprevalence of M. bovis was 26.6 per cent on day 0 and 98.2 per cent by day 42 (P<0.05). Seroconversion to M. bovis between days 0 and 42 was significantly associated (P=0.04) with lower weight gain. Weight gain was greater in calves that were PCR-negative for M. bovis on day 10 (P=0.01). The percentage of calves seropositive to M. bovis increased throughout the study, indicating exposure and an immunological response to the organism. Although associations with health outcomes were not identified, seroconversion to M. bovis was associated with a decreased rate of weight gain during the study period.

Bovine coronavirus (BCV) infections in transported commingled beef cattle and sole-source ranch calves.

This study investigated bovine coronavirus (BCV) in both beef calves direct from the ranch and commingled, mixed-source calves obtained from an auction market. The level of BCV-neutralizing antibodies found in the calves varied among ranches in 2 different studies in a retained-ownership program (ROP), from the ranch to the feedlot. Calves with low levels of BCV-neutralizing antibodies (16 or less) were more likely to be treated for bovine respiratory disease (BRD) than those with higher titers. In 3 studies of commingled, mixed-source calves, BCV was recovered from calves at entry to the feedlot and the infections were cleared by day 8. The BCV was identified in lung samples [bronchoalveolar lavage (BAL) collection] as well as in nasal swabs. Calves with low levels of BCV-neutralizing antibodies at entry were most likely to be shedding BCV. Bovine coronavirus was isolated from both healthy and sick calves, but not from sick calves after 4 d arrival at the feedlot. Bovine coronavirus (BCV) should be considered along with other bovine respiratory viruses in the diagnosis of etiologies in bovine respiratory disease, especially for animals that become sick shortly after arrival. If approved vaccines are developed, it would be best to carry out vaccination programs before calves are weaned, giving them sufficient time to gain active immunity before commingling with other cattle.

Plasma metabolites of receiving heifers and the relationship between apparent bovine respiratory disease, body weight gain, and carcass characteristics.

Six hundred sixty-five crossbred beef heifers initially weighing 225 kg were used in a completely randomized design to measure plasma glucose, lactate, and urea N concentrations at time of initial processing, determine the incidence of apparent bovine respiratory disease (BRD) in receiving cattle, and evaluate the effect of apparent BRD on subsequent cattle growth and carcass characteristics. Heifers were processed within 24 h of arrival, and processing included vaccination against common viral and clostridial diseases, recording rectal temperature, and sampling whole blood for subsequent measurement of plasma glucose, lactate, and urea concentrations. Heifers were monitored for clinical signs of apparent BRD, including depression, lethargy, anorexia, coughing, rapid breathing, and nasal or ocular discharge. Heifers exhibiting signs of apparent BRD received antibiotic therapy, and the number of times a heifer was treated for apparent BRD was recorded. Following the 36-d receiving period, heifers were transported to native grass pastures and allowed to graze for 136 d. At the end of the grazing season, heifers were transported to a commercial feedlot where they were adapted to a common finishing diet offered for ad libitum consumption. Following the 124-d finishing period, heifers were slaughtered and carcass data were collected. Heifers treated for apparent BRD had decreased plasma glucose (linear, P < 0.01), lactate (linear, P < 0.01), and urea N concentrations (linear, P < 0.06) measured at time of initial processing. Rectal temperature measured at time of initial processing tended to be greater (linear, P < 0.11) for heifers treated for apparent BRD. Heifers treated for apparent BRD during the receiving period had decreased overall ADG (linear, P < 0.10), final BW (linear, P < 0.01), HCW (linear, P < 0.01), fat thickness (linear, P < 0.01), and marbling score (linear, P < 0.03). These data suggest that initial plasma glucose and lactate concentrations might be affected by the health status of receiving cattle and that increased incidence of apparent BRD in cattle decreases ADG and carcass quality.

Comparative approaches to the investigation of responses to stress and viral infection in cattle.

Fatal bovine respiratory disease (BRD) is a major cause of financial losses in the cattle industry. A variety of stressors have been implicated as contributing to disease severity. However, it has proven difficult to determine the role these individual factors may play in the final outcome of this disease complex. The objective of the present investigation was to obtain proteomic, metabonomic, and elemental profiles of bovine serum samples from stressed and control animals before and after a primary viral infection to determine if these profiles could distinguish between responses to stressors and viral infection. Multivariate analysis revealed distinct differential trends in the distribution profile of proteins, metabolites, and elements following a stress response both before and after primary viral infection. A group of acute phase proteins, metabolites, and elements could be specifically linked to either a stress response (decreased serum amyloid A and Cu, increased apolipoprotein CIII, amino acids, LDL, P, and Mo) or a primary viral respiratory infection (increased apolipoprotein A1, haptoglobin, glucose, amino acids, LDL and Cu, decreased Lipid, and P). Thus, combined OMICS analysis of serum samples revealed that multimethod analysis could be used to discriminate between the complex biological responses to stress and viral infection.