Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis.

Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.

Pharmacokinetic report: Pharmacokinetics of a single oral dose of gabapentin in goats (Capra hircus).

Gabapentin is used in goats to treat chronic pain associated with lameness. However, pharmacokinetic data and clinical effectiveness trials are lacking. The objective of the study was to describe the pharmacokinetics of gabapentin in goats following a single oral dose. Six Spanish-crossbred goats were enrolled. Each goat was administered gabapentin at a target dose of 15 mg/kg per os. Serial blood samples were collected out to 60 h post-gabapentin administration for plasma gabapentin concentration determination. Plasma samples were analyzed for gabapentin concentration using ultra-high-pressure liquid chromatography coupled with mass spectroscopy. Individual animal pharmacokinetic outcomes were determined using non-compartmental analysis. Gabapentin was detectable in the plasma of all goats at 60 h post-administration. The mean (±SD) Cmax was 2.01 ± 0.62 µg/mL which occurred at 8.47 ± 1.9 h. The mean terminal half-life (T1/2) and mean resident time were determined to be 8.52 ± 1.8 and 18.7 ± 4.0 h, respectively. This study indicates gabapentin is absorbed from the gastrointestinal tract of goats. Further research is needed to determine an optimal dose for clinical efficacy in goats.

Effect of bupivacaine liposome suspension administered as a cornual nerve block on indicators of pain and distress during and after cautery dehorning in dairy calves.

Dehorning is performed on a high percentage of dairies worldwide. Concern about the negative effect of dehorning on animal welfare has contributed to the development of new guidelines that require the use of pain management at the time of disbudding in the United States. However, livestock producers are limited in how to address this requirement due to a lack of (1) approved analgesic drugs, (2) analgesic options that control pain for an extended duration, and (3) analgesic formulations that are practical for producers to administer. The objective of this study was to evaluate the effectiveness of bupivacaine liposome suspension, a novel, long-acting, local anesthetic formulation administered as a nerve block at dehorning, compared with current industry standard analgesic approaches using lidocaine nerve blocks alone or in combination with the nonsteroidal anti-inflammatory drug meloxicam. Fifty male Holstein calves, 10 to 14 wk of age, were enrolled and randomly assigned to 1 of 5 treatment groups before cautery dehorning as follows: (1) bupivacaine liposome suspension block, oral placebo (BUP); (2) lidocaine block, oral placebo (LID); (3) lidocaine block, oral meloxicam (1 mg/kg of body weight; LID + MEL); (4) saline block, oral placebo (CON); and (5) saline block, oral placebo, sham dehorn (SHAM). Biomarkers were collected from 0 to 120 h postdehorning and included infrared thermography, mechanical nociceptive threshold (MNT), pressure mat gait analysis, chute defense and behavior scoring, and blood sampling for serum cortisol and prostaglandin E2 metabolites. Responses were analyzed using repeated measures with calf nested in treatment designated as a random effect, and treatment, time, and their interaction designated as fixed effects. At 2 h postdehorning, the BUP group had a higher MNT compared with the CON group. Furthermore, at 24 h postdehorning, the BUP group had a higher MNT compared with the LID group. Gait distance differed significantly between treatment groups; the CON, LID, and LID + MEL groups had an increased gait distance relative to the SHAM group. The CON group exhibited a higher chute defense behavior score during the dehorning procedure compared with all other treatments. Furthermore, the CON group exhibited more ear flicks than the BUP and LID + MEL groups postdehorning. At 4 h and 24 h after dehorning, the LID + MEL group had a lower average prostaglandin E2 metabolites concentration compared with all other treatment groups. These data showed that administration of bupivacaine liposome suspension as a cornual nerve block at the time of dehorning was as effective at controlling pain as a multimodal approach of lidocaine and meloxicam.

Pharmacokinetics and tissue concentrations of firocoxib in sows following oral administration.

The objectives of this study were to describe the pharmacokinetics of firocoxib following oral (PO) dosing and intravenous (IV) injection in sows. Seven healthy sows were administered 0.5 mg firocoxib/kg IV. Following a 23-d washout period, sows were administered firocoxib at 4.0 mg firocoxib/kg PO. Blood samples were collected at predetermined times for 72 hr after IV and 120 hr after PO administration. Plasma firocoxib concentration was measured using UPLC-MS/MS, and pharmacokinetic analysis was performed using noncompartmental procedures. Tissue firocoxib concentrations were determined at 5, 10 (n = 2/time point), and 21 d (n = 3) after PO administration. The geometric mean half-life following IV and PO administration was 16.6 and 22.5 hr, respectively. A mean peak plasma concentration (Cmax) of 0.06 µg/ml was recorded at 7.41 hr (Tmax ) after oral administration. Mean oral bioavailability was determined to be 70.3%. No signs of NSAID toxicity were observed on macroscopic and microscopic investigation. Firocoxib was detected in the skin with subcutaneous fat (0.02 µg/g) of one of three sows at 21 days postadministration. Additional work to establish appropriate meat withhold intervals in sows is required. Firocoxib was readily absorbed following PO administration. Further work is needed to better understand the analgesic effects for sows and piglets nursing sows administered firocoxib.

Pharmacokinetics and pharmacodynamics of intravenous and transdermal flunixin meglumine in meat goats.

The aim of this study was to determine the pharmacokinetics and prostaglandin E2 (PGE2 ) synthesis inhibiting effects of intravenous (IV) and transdermal (TD) flunixin meglumine in eight adult female Boer goats. A dose of 2.2 mg/kg was administered intravenously (IV) and 3.3 mg/kg administered TD using a cross-over design. Plasma flunixin concentrations were measured by LC-MS/MS. Prostaglandin E2 concentrations were determined using a commercially available ELISA. Pharmacokinetic (PK) analysis was performed using noncompartmental methods. Plasma PGE2 concentrations decreased after flunixin meglumine for both routes of administration. Mean λz -HL after IV administration was 6.032 hr (range 4.735-9.244 hr) resulting from a mean Vz of 584.1 ml/kg (range, 357.1-1,092 ml/kg) and plasma clearance of 67.11 ml kg-1  hr-1 (range, 45.57-82.35 ml kg-1  hr-1 ). The mean Cmax , Tmax, and λz -HL for flunixin following TD administration was 0.134 µg/ml (range, 0.050-0.188 µg/ml), 11.41 hr (range, 6.00-36.00 hr), and 43.12 hr (15.98-62.49 hr), respectively. The mean bioavailability for TD flunixin was calculated as 24.76%. The mean 80% inhibitory concentration (IC80 ) of PGE2 by flunixin meglumine was 0.28 µg/ml (range, 0.08-0.69 µg/ml) and was only achieved with IV formulation of flunixin in this study. The PK results support clinical studies to examine the efficacy of TD flunixin in goats. Determining the systemic effects of flunixin-mediated PGE2 suppression in goats is also warranted.

Pharmacokinetics and pharmacodynamics of intravenous and transdermal flunixin meglumine in alpacas.

The aim of this study was to determine the pharmacokinetics and prostaglandin E2 (PGE2 ) synthesis inhibiting effects of intravenous (IV) and transdermal (TD) flunixin meglumine in eight, adult, female, Huacaya alpacas. A dose of 2.2 mg/kg administered IV and 3.3 mg/kg administered TD using a cross-over design. Plasma flunixin concentrations were measured by LC-MS/MS. Prostaglandin E2 concentrations were determined using a commercially available ELISA. Pharmacokinetic (PK) analysis was performed using noncompartmental methods. Plasma PGE2 concentrations decreased after IV flunixin meglumine administration but there was minimal change after TD application. Mean t1/2 λz after IV administration was 4.531 hr (range 3.355 to 5.571 hr) resulting from a mean Vz of 570.6 ml/kg (range, 387.3 to 1,142 ml/kg) and plasma clearance of 87.26 ml kg-1  hr-1 (range, 55.45-179.3 ml kg-1  hr-1 ). The mean Cmax, Tmax and t1/2 λz for flunixin following TD administration were 106.4 ng/ml (range, 56.98 to 168.6 ng/ml), 13.57 hr (range, 6.000-34.00 hr) and 24.06 hr (18.63 to 39.5 hr), respectively. The mean bioavailability for TD flunixin was calculated as 25.05%. The mean 80% inhibitory concentration (IC80 ) of PGE2 by flunixin meglumine was 0.23 µg/ml (range, 0.01 to 1.38 µg/ml). Poor bioavailability and poor suppression of PGE2 identified in this study indicate that TD flunixin meglumine administered at 3.3 mg/kg is not recommended for use in alpacas.

Comparative plasma and interstitial fluid pharmacokinetics and tissue residues of ceftiofur crystalline-free acid in cattle with induced coliform mastitis.

Ceftiofur (CEF) is a third-generation cephalosporin that is the most widely used antimicrobial in the dairy industry. Currently, violative meat residues in cull dairy cattle are commonly associated with CEF. One potential cause for violative residues is altered pharmacokinetics of the drug due to disease, which could increase the time needed for the residue to deplete. The objectives of this study were (a) to determine the absolute bioavailability of CEF crystalline-free acid (CFA) in healthy versus diseased cows; (b) to compare the plasma and interstitial fluid pharmacokinetics and plasma protein binding of CEF between healthy dairy cows and those with disease; and (c) to determine the CEF residue profile in tissues of diseased cows. For this trial, disease was induced through intramammary Escherichia coli infusion. Following disease induction and CEF CFA administration, for plasma concentrations, there was not a significant effect of treatment (p = 0.068), but the treatment-by-time interaction (p = 0.005) was significant. There was a significantly greater concentration of CEF in the plasma of the DIS cows at T2 hr (p = 0.002), T8 hr (p < 0.001), T12 hr (p = 0.001), and T16 hr (p = 0.002). For PK parameters in plasma, the slope of the terminal phase of the concentration versus time curve was significantly lower (p = 0.007), terminal half-life was significantly longer (p = 0.014), and apparent volume of distribution during the elimination phase was significantly higher (p = 0.028) diseased group. There was no difference in plasma protein binding of CEF and interstitial fluid pharmacokinetics. None of the cows had kidney CEF residues above the US tolerance level following observation of the drug's withdrawal period, but one cow with a larger apparent volume of distribution and longer terminal half-life had tissue residues slightly below the tolerance. Whereas these findings do not support the hypothesis that severely ill cows need longer withdrawal times, alterations in the terminal half-life suggest that it is theoretically possible.

The impact of pain on the pharmacokinetics of transdermal flunixin meglumine administered at the time of cautery dehorning in Holstein calves.

OBJECTIVE: To study the influence of pain on the pharmacokinetics and anti-inflammatory actions of transdermal flunixin administered at dehorning. STUDY DESIGN: Prospective, crossover, clinical study. ANIMALS: A total of 16 male Holstein calves, aged 6-8 weeks weighing 61.3 ± 6.6 kg. METHODS: Calves were randomly assigned to one of two treatments: transdermal flunixin and dehorning (PAIN) or transdermal flunixin and sham dehorning (NO PAIN). Flunixin meglumine (3.33 mg kg-1) was administered topically as a pour-on concurrently with hot iron dehorning or sham dehorning. The calves were subjected to the alternative treatment 14 days later. Blood samples were collected at predetermined time points up to 72 hours for measurement of plasma flunixin concentrations. Pharmacokinetics parameters were determined using noncompartmental analysis. Prostaglandin E2 (PGE2) concentration was determined using a commercial enzyme-linked immunosorbent assay. The 80% inhibition concentration (IC80) of PGE2 was determined using nonlinear regression. Pharmacokinetic data were statistically analyzed using paired t tests and Wilcoxon rank sums for nonparametric data. Flunixin and PGE2 concentrations were log transformed and analyzed using repeated measures. RESULTS: A total of 15 calves completed the study. Plasma half-life of flunixin was significantly longer in PAIN (10.09 hours) than NO PAIN (7.16 hours) (p = 0.0202). Bioavailability of transdermal flunixin was 30% and 37% in PAIN and NO PAIN, respectively (p = 0.097). Maximum plasma concentrations of flunixin were 0.95 and 1.16 µg mL-1 in PAIN and NO PAIN, respectively (p = 0.089). However, there was a treatment (PAIN versus NO PAIN) by time interaction (p = 0.0353). PGE2 concentrations were significantly lower in the PAIN treatment at 48 and 72 hours (p = 0.0092 and p = 0.0287, respectively). The IC80 of PGE2 by flunixin was similar in both treatments (p = 0.88). CONCLUSION AND CLINICAL RELEVANCE: Pain alters the pharmacokinetics and anti-inflammatory effects of transdermally administered flunixin.

Off-Target drug effects resulting in altered gene expression events with epigenetic and "Quasi-Epigenetic" origins.

This review synthesizes examples of pharmacological agents who have off-target effects of an epigenetic nature. We expand upon the paradigm of epigenetics to include "quasi-epigenetic" mechanisms. Quasi-epigenetics includes mechanisms of drugs acting upstream of epigenetic machinery or may themselves impact transcription factor regulation on a more global scale. We explore these avenues with four examples of conventional pharmaceuticals and their unintended, but not necessarily adverse, biological effects. The quasi-epigenetic drugs identified in this review include the use of beta-lactam antibiotics to alter glutamate receptor activity and the action of cyclosporine on multiple transcription factors. In addition, we report on more canonical epigenome changes associated with pharmacological agents such as lithium impacting autophagy of aberrant proteins, and opioid drugs whose chronic use increases the expression of genes associated with addictive phenotypes. By expanding our appreciation of transcriptomic regulation and the effects these drugs have on the epigenome, it is possible to enhance therapeutic applications by exploiting off-target effects and even repurposing established pharmaceuticals. That is, exploration of "pharmacoepigenetic" mechanisms can expand the breadth of the useful activity of a drug beyond the traditional drug targets such as receptors and enzymes.

Genetically modified live vaccine offers protective immunity against wild-type Anaplasma marginale tick-transmission challenge.

Anaplasma marginale is a tick-borne pathogen of cattle that causes bovine anaplasmosis in tropical and subtropical regions throughout the world. Killed vaccines derived from infected erythrocytes have been used for control of this disease with limited success. Recently, we described a targeted deletion mutation in the phage head-to-tail connector protein gene of A. marginale which caused bacterial attenuation in vivo and provided protection as a modified live vaccine (MLAV). Following intravenous injection of susceptible steers, the MLAV induced protective immunity against disease progression. In the current study, we demonstrated that the immunity resulting from MLAV in cattle prevents the disease progression resulting from virulent A. marginale intrastadial transmission from infected Dermacentor variabilis male ticks. The nonimmunized control steers receiving the infection from ticks developed fever, lethargy, and inappetence for several days post tick exposure with significant decreases in the packed cell volume and increases in bacteremia. In contrast, the MLAV immunized steers remained healthy after being challenged with infected ticks and this group of animals had a significant reduction in bacteremia as compared with the controls. This study demonstrated that the A. marginale MLAV provided protection against acute tick-transmitted anaplasmosis, in addition to protection documented in steers challenge-exposed with infected blood as reported previously.

Comparison of firocoxib and meloxicam for pain mitigation in goats undergoing surgical castration.

OBJECTIVE: This study sought to determine whether firocoxib (FIRO) or meloxicam (MEL) was effective at providing analgesia after surgical castration in goats. ANIMALS: 18 intact male crossbred goats (6 to 8 months old) were enrolled with a mean weight of 32.6 (± 2.9) kg. METHODS: Surgical castration was done under injectable anesthesia by a licensed veterinarian. Twelve bucks were surgically castrated and given either FIRO (n = 6) or MEL (n = 6). Six bucks served as controls (CNTLs) and were not castrated. Outcome measurements included visual analogue scale, infrared thermography, plasma cortisol, plasma substance P, and kinetic gait analysis. All outcome measurements were obtained at -24, 4, 8, 24, 48, and 72 hours. RESULTS: All 3 treatments were significantly different from each other at the 24- and 48-hour time points, with MEL animals having lower visual analogue scale scores when compared to FIRO animals; CNTL animals exhibited the lowest plasma cortisol levels (3.19 ng/mL; 95% CI, -1.21 to 7.59 ng/mL) followed by FIRO (7.45 ng/mL; 95% CI, 3.10 to 11.80 ng/mL) and MEL (10.24 ng/mL; 95% CI, 5.87 to 14.60 ng/mL). FIRO had an average mean decrease in gait velocity change (-54.17 cm/s; 95% CI, -92.99 to -15.35 cm/s), while MEL had an increase in gait velocity when compared to baseline values (14.54 cm/s; 95% CI, -24.27 to 53.36 cm/s). Control animals had an average mean of -3.06 cm/s (95% CI, -41.88 to 35.75 cm/s). CLINICAL RELEVANCE: Results from this study showed that there were some analgesic effects from administering MEL when compared to bucks that received a placebo treatment (CNTL).

Measurement of Cyclooxygenase Products in Plasma as Markers for Inhibition of Cyclooxygenase Isoforms by Oral Meloxicam in New Zealand White Rabbits (Oryctolagus cuniculus ).

Pain management in rabbits is a challenging task that is complicated by the rabbit's ability to hide signs of distress and the limited pharmacologic data available for this species. Pharmacokinetic data has shown that in rabbits, meloxicam, a nonsteroidal anti-inflammatory NSAID, reaches plasma concentrations that are known to provide analgesia in dogs and cats; these concentrations could theoretically alleviate pain in rabbits. However, the inhibitory effects of meloxicam on cyclooxygenase (COX) isoforms have not been studied in rabbits. In this study, we measured the products of COX-1 and COX-2 after the oral administration of a single 1 mg/kg dose of meloxicam to New Zealand White rabbits (n = 6). Blood samples were collected before drug administration (T0) and then at predetermined time points over 48 h. Plasma prostaglandin E2 (PGE2 ) and thromboxane (TxB2) concentrations were measured as surrogate markers for COX-1 and COX-2, respectively, by using commercial ELISA kits. After meloxicam administration, both TxB2 and PGE2 plasma concentrations fell significantly below baseline, with maximal mean reductions to 80% and 60% of baseline at 8 h, respectively. The reduction in PGE2 concentrations was followed by a significant increase that moved its mean plasma concentrations toward baseline between 8 and 24 h. Adverse effects such as lethargy, inappetence, or changes in fecal production were not observed in any rabbits. In conclusion, meloxicam appeared to significantly inhibit both COX-1 and COX-2 with a time course similar to previously reported meloxicam plasma concentration-time profiles in rabbits. Our data suggest that a dosage of 1 mg/kg given orally could provide analgesia to rabbits, but a more frequent dosing interval than the currently recommended daily dosing may be required to maintain clinical efficacy.

Pharmacokinetics, Milk Residues, and Toxicological Evaluation of a Single High Dose of Meloxicam Administered at 30 mg/kg per os to Lactating Dairy Cattle.

Adverse effects associated with overdose of NSAIDs are rarely reported in cattle, and the risk level is unknown. If high doses of NSAIDs can be safely administered to cattle, this may provide a longer duration of analgesia than using current doses where repeated administration is not practical. Meloxicam was administered to 5 mid-lactation Holstein dairy cows orally at 30 mg/kg, which is 30 times higher than the recommended 1 mg/kg oral dose. Plasma and milk meloxicam concentrations were determined using high-pressure liquid chromatography with mass spectroscopy (HPLC-MS). Pharmacokinetic analysis was performed by using noncompartmental analysis. The geometric mean maximum plasma concentration (Cmax) was 91.06 µg/mL at 19.71 h (Tmax), and the terminal elimination half-life (T1/2) was 13.79 h. The geometric mean maximum milk concentration was 33.43 µg/mL at 23.74 h, with a terminal elimination half-life of 12.23 h. A thorough investigation into the potential adverse effects of a meloxicam overdose was performed, with no significant abnormalities reported. The cows were humanely euthanized at 10 d after the treatment, and no gross or histologic lesions were identified. As expected, significantly higher plasma and milk concentrations were attained after the administration of 30 mg/kg meloxicam with similar half-lives to previously published reports. However, no identifiable adverse effects were observed with a drug dose 30 times greater than the industry uses within 10 days of treatment. More research is needed to determine the tissue withdrawal period, safety, and efficacy of meloxicam after a dose of this magnitude in dairy cattle.

Optimal lameness induction model development using amphotericin B in meat goats.

Lameness continues to be a critical health and welfare concern associated with goat production. Amphotericin B (amp B) is an antimicrobial successful in inducing transient lameness for research purposes previously in livestock animals. The objectives of this study were to (1) identify which of three varying doses of amp B would be most effective in inducing lameness in meat type goats and (2) develop a facial grimace scale for goats. Lameness was produced by an intra-articular injection of amphotericin B into the left hind lateral claw distal interphalangeal joint with either a 5 mg/0.25 mL (high-low, 5 mg of amphotericin B in a volume of 0.25 mL), 5 mg/0.5 mL (high-high, 5 mg of amphotericin B in a volume of 0.5 mL), or a 2.5 mg/0.25 mL (low-low, 2.5 mg of amphotericin B in a volume of 0.25 mL). A saline treatment of 0.5 mL was used as control (0.9% sterile saline solution). Lameness response was analyzed by infrared thermography (IRT) at the induced joint, mechanical-nociception threshold (MNT), visual lameness scoring (VLS), a visual analogue scale (VAS), kinetic gait analysis (KGA), plasma cortisol (CORT), substance P (Sub P), and behavior scoring. The IRT and MNT values differed by timepoint (P ≤ 0.0001). Results from VLS showed the HL treatment was the most effective at inducing lameness (6/6 goats became lame compared to HH 4/6 and LL 2/6). At 24, 48, and 72 h, VAS scores were significantly higher when comparing HL to all other treatment groups (P = 0.0003). Both behavior observers (1 and 2) reported a significant time effect (P = 0.05), with goats exhibiting more facial grimacing at 24 h post-lameness induction. From these data, an optimal dose for a repeatable lameness induction model in goats was aquired. An effective Goat Grimace Scale (GGS) was also developed to evaluate pain responses in goats.

Assessment of diagnostic accuracy of biomarkers to assess lung consolidation in calves with induced bacterial pneumonia using receiver operating characteristic curves.

Bovine respiratory disease (BRD) is the most economically significant disease for cattle producers in the U.S. Cattle with advanced lung lesions at harvest have reduced average daily gain, yield grades, and carcass quality outcomes. The identification of biomarkers and clinical signs that accurately predict lung lesions could benefit livestock producers in determining a BRD prognosis. Receiver operating characteristic (ROC) curves are graphical plots that illustrate the diagnostic ability of a biomarker or clinical sign. Previously we used the area under the ROC curve (AUC) to identify cortisol, hair cortisol, and infrared thermography imaging as having acceptable (AUC > 0.7) diagnostic accuracy for detecting pain in cattle. Herein, we used ROC curves to assess the sensitivity and specificity of biomarkers and clinical signs associated with lung lesions after experimentally induced BRD. We hypothesized pain biomarkers and clinical signs assessed at specific time points after induction of BRD could be used to predict lung consolidation at necropsy. Lung consolidation of > 10% was retrospectively assigned at necropsy as a true positive indicator of BRD. Calves with a score of < 10% were considered negative for BRD. The biomarkers and clinical signs analyzed were serum cortisol; infrared thermography (IRT); mechanical nociceptive threshold (MNT); substance P; kinematic gait analysis; a visual analog scale (VAS); clinical illness score (CIS); computerized lung score (CLS); average activity levels; prostaglandin E2 metabolite (PGEM); serum amyloid A; and rectal temperature. A total of 5,122 biomarkers and clinical signs were collected from 26 calves, of which 18 were inoculated with M. haemolytica. All statistics were performed using JMP Pro 14.0. Results comparing calves with significant lung lesions to those without yielded the best diagnostic accuracy (AUC > 0.75) for right front stride length at 0 h; gait velocity at 32 h; VAS, CIS, average activity and rumination levels, step count, and rectal temperature, all at 48 h; PGEM at 72 h; gait distance at 120 h; cortisol at 168 h; and IRT, right front force and serum amyloid A, all at 192 h. These results show ROC analysis can be a useful indicator of the predictive value of pain biomarkers and clinical signs in cattle with induced bacterial pneumonia. AUC values for VAS score, average activity levels, step count, and rectal temperature seemed to yield good diagnostic accuracy (AUC > 0.75) at multiple time points, while MNT values, substance P concentrations, and CLS did not (all AUC values < 0.75).

Bovine respiratory disease (BRD) is the most economically significant disease for cattle producers in the United States, affecting 16.2% of cattle on feed. Cattle with advanced lung lesions at harvest have reduced average daily gain, yield grades, and carcass quality outcomes. The identification of biomarkers and clinical signs that accurately predict lung lesions could benefit livestock producers in determining a BRD prognosis. Herein, we used receiver operating characteristic curves to assess the predictive value of biomarkers and clinical signs associated with lung lesions after experimentally induced BRD. In the first 72 h after onset of BRD, right front stride length, gait velocity, visual analog scale score, clinical illness score, average activity level, step count, and rectal temperature yielded the best diagnostic accuracy (AUC > 0.75) for predicting calves with significant lung lesions (>10% consolidation) at necropsy. Biomarkers and clinical signs with the best diagnostic accuracy early in the disease process would likely be the most valuable in field conditions. These results can be used to guide refinement of the optimal time points and biomarkers for the diagnosis of significant lung lesions after BRD.

Short term feeding of industrial hemp with a high cannabidiolic acid (CBDA) content increases lying behavior and reduces biomarkers of stress and inflammation in Holstein steers.

Industrial hemp (IH) is defined as Cannabis sativa containing < 0.3% delta-9 tetrahydrocannabinol (THC) and was legalized in the 2018 Farm Bill. The impact of cannabinoids in IH fed to livestock, especially after repeat exposure, has not been thoroughly investigated. Sixteen male castrated Holstein cattle weighting (± SD) 447 ± 68 kg were enrolled onto the study. Cattle were allocated into two treatment groups either receiving IH (HEMP, n = 8) or a control (CNTL, n = 8). Cattle in the HEMP group were fed 25 g IH mixed in 200 g of grain once a day for 14 days to target a daily dose of 5.5 mg/kg of cannabidiolic acid (CBDA). Behavior was continuously monitored with accelerometers and blood samples were collected at predetermined time points for plasma cannabinoid, serum cortisol, serum haptoglobin, liver enzymes, serum amyloid A, and prostaglandin E2 concentrations. The HEMP group spent a mean 14.1 h/d (95% CI 13.6-14.6 h/d) lying compared to the 13.4 h/d (95% CI 12.9-13.8 h/d) for the CNTL cattle (P = 0.03). Cortisol concentrations in the HEMP group were lower than the CNTL group (P = 0.001). Cattle in the HEMP group demonstrated an 8.8% reduction in prostaglandin E2 concentrations from baseline compared to a 10.2% increase from baseline observed in the CNTL group. No differences for haptoglobin or serum amyloid A were observed. These results suggest that feeding IH with a high CBDA content for 14 days increases lying behavior and decreases biomarkers of stress and inflammation in cattle.

Rapid quantification of cannabinoids in beef tissues and bodily fluids using direct-delivery electrospray ionization mass spectrometry.

Hempseed cake is a byproduct of hempseed oil extraction and is potentially a useful source of protein and fiber for use in ruminant diets. However, data are lacking on the appearance and/or clearance of cannabinoids in tissues of animals fed hempseed cake. To this end, a rapid method for quantifying cannabinol (CBN), cannabidiol (CBD), cannabinolic acid (CBNA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabinol (THC) and tetrahydrocannabinolic acid (THCA) in cattle tissues, plasma, and urine was developed using rapid screen electrospray ionization mass spectrometry (RS-ESI-MS). Regression coefficients of matrix-matched standard curves ranged from 0.9946 to >0.9999 and analyte recoveries averaged from 90.2 ± 15.5 to 108.7 ± 18.7% across all compounds. Limits of detection and quantification ranged from 0.05 to 2.79 ng · mL-1 and 0.17 to 9.30 ng · mL-1, respectively, while the inter-day relative standard deviation ranged from 5.1 to 15.1%. Rapid screening electrospray ionization mass spectrometry (RS-ESI-MS) returned no false positives for any cannabinoid in plasma, urine, and tissue (liver, skeletal muscle) samples from 6 non-dosed control animals (n = 90 samples; of which 72 samples were plasma or urine and 18 samples were tissues). Across-animal cannabinoid concentrations measured in 32 plasma samples of cattle dosed with ground hemp were quantified by RS-ESI-MS; analytical results correlated well (r2 = 0.963) with independent LC-MS/MS analysis of the same samples.

Comparison of lidocaine alone or in combination with a local nerve block of ethanol, bupivacaine liposome suspension, or oral meloxicam to extend analgesia after scoop dehorning in Holstein calves.

The American Veterinary Medical Association recommends the use of practices that reduce or eliminate pain and discomfort associated with dehorning. Identification of an effective, long-acting local anesthetic that is practical for producers to implement and reduces pain from dehorning would benefit animal welfare. Thirty-two Holstein bulls and heifers were enrolled. The objective of this study was to compare the efficacy and duration of activity of bupivacaine liposome suspension (BUP; n = 8), ethanol (ETH; n = 8), or meloxicam (LID + MEL; n = 8) co-administered with lidocaine compared with lidocaine only (LID; n = 8), and to quantify their effect on pain biomarkers and behaviors after scoop dehorning with cauterization in approximately 20-wk-old calves. Outcome variables collected included infrared thermography (IRT), mechanical nociceptive threshold (MNT), visual analog scale (VAS) scoring, and blood sampling for serum cortisol and prostaglandin E2 metabolites (PGEM). There was evidence of a sex effect for MNT, with bulls demonstrating a higher threshold (13.74 kgf) compared with heifers (12.12 kgf). There was a treatment by time interaction for cortisol concentrations (ng/mL). At 2 h, the BUP group had higher cortisol values (17.32 ng/mL) than the LID + MEL group (3.10 ng/mL). Heifers also had higher mean cortisol values (13.88 ng/mL) compared with bulls (6.96 ng/mL). There was a treatment by time interaction for PGEM concentration. Calves in the LID + MEL group had lower PGEM values at 4 and 8 h (10.23 and 9.12 pg/mL) than at -24, 0, and 0.5 h (20.38, 27.27, and 22.59 pg/mL, respectively). At 4 h, the LID + MEL group had lower PGEM concentrations (10.23 pg/mL) than the ETH group (27.08 pg/mL). At 8 h, the LID + MEL group had lower PGEM concentrations (9.12 pg/mL) than both the ETH and BUP groups (24.80 and 20.52 pg/mL). Thus, LID + MEL reduced cortisol and prostaglandin metabolite concentrations more effectively than ETH + LID or BUP + LID administered as a local infiltration and cornual block, respectively, before scoop dehorning followed by cauterization. The treatments administered in the present study did not seem to extend the duration of analgesia beyond the currently recommended multimodal approach, including local anesthesia and systemic analgesia such as lidocaine and meloxicam. Evidence from the current study suggests that sex influences pain biomarkers such as nociceptive threshold and cortisol concentration, with males having a higher nociceptive threshold and lower cortisol responses.

Behavioral and performance response associated with administration of intravenous flunixin meglumine or oral meloxicam immediately prior to surgical castration in bull calves.

The objective of this study was to determine the effects of flunixin meglumine or meloxicam on behavioral response and performance characteristics associated with surgical castration in crossbred bulls. Intact male Bos taurus calves (n = 252; averaging 176 kg) were randomly allocated into one of three treatment groups within pen: control (CON), flunixin meglumine (FLU; 2.2 mg/kg intravenous injection), or meloxicam (MEL; 2.0 mg/kg per os). The individual animal was the experimental unit. Calves were individually weighed on days 0 and 14 of the trial to evaluate performance outcomes. On study day 0, treatments were administered, according to their random allocation, immediately prior to surgical castration using the Henderson tool method. Visual analog scale (VAS) assessment and categorical attitude score (CAS) were collected on days -1, 0 (6 h post-castration), 1, 2, 3, and 4 in the study. The VAS was assigned using a 100 mm horizontal line with "normal" labeled at one end of the line and "moribund" at the other end of the horizontal line. The masked observer assigned a mark on the horizontal line based upon the observed severity of pain exhibited by that individual animal. The CAS was assigned by the same observer using five different categories with a score of 0 being "normal". Average daily gain tended (P = 0.09) to be associated with the treatment group, and MEL had a greater (P = 0.04) average daily gain through day 14 compared with CON. A significant (P < 0.01) treatment by day interaction was indicated for VAS score, and MEL had lower VAS scores on days 0, 1, 2, and 3 post-castration compared with CON; FLU had lower VAS scores on days 0 and 1 compared with CON. A significant treatment by day interaction was not present (P = 0.25) for CAS. The FLU had lesser percent CAS ≥1 (17.5%; P = 0.05) compared with CON (29.4%); MEL has lesser percent CAS ≥1 observations (14.9%; P = 0.01) compared with CON. The median VAS increased as CAS was more severe. Results indicated MEL and FLU calves temporally improved behavioral responses following surgical castration with positive numerical trends for a 14 d average daily gain (ADG). The VAS system appeared to be an effective method of subjective evaluation of pain in beef calves in this study. Route of administration, duration of therapy, and low relative cost make oral meloxicam a reasonable analgesic treatment in calves when administered at the time of surgical castration.

The effect of breed, sex, and oral meloxicam administration on pain biomarkers following hot-iron branding in Hereford and Angus calves.

Hot-iron branding uses thermal injury to permanently identify cattle causing painful tissue damage. The primary objective was to examine the physiological and behavioral effects of oral meloxicam (MEL), compared to a control, administered at the time of hot-iron branding in Angus and Hereford steers and heifers. The secondary objectives were to investigate breed and sex effects on pain biomarkers. A total of 70 yearlings, consisting of 35 heifers and 35 steers (Angus, Hereford, or Angus × Hereford), were enrolled in the study. Animals were blocked by sex, randomized across weight, and assigned to receive MEL (1 mg/kg) or a placebo (CON). Biomarkers were assessed for 48 h after branding and included infrared thermography (IRT), mechanical nociceptive threshold (MNT), accelerometry and a visual analog scale (VAS), and serum cortisol and prostaglandin E2 metabolites (PGEM). Wound healing was assessed for 12 wk. Hair samples to quantify cortisol levels were taken prior to and 30 d post-branding. Responses were analyzed using repeated measures with calf nested in treatment as a random effect, and treatment, time, treatment by time interaction, breed, and sex as fixed effects. There was a treatment by time interaction for PGEM (P < 0.01) with MEL having lower values than CON at 6, 24, and 48 h (MEL: 18.34 ± 3.52, 19.61 ± 3.48, and 22.24 ± 3.48 pg/mL, respectively; CON: 32.57 ± 3.58, 37.00 ± 3.52, and 33.07 ± 3.48 pg/mL; P < 0.01). MEL showed less of a difference in maximum IRT values between the branded (2.27 ± 0.29 °C) and control site (3.15 ± 0.29 °C; P < 0.01). MEL took fewer lying bouts at 0-12 h (4.91 bouts ± 0.56) compared with CON (6.87 bouts ± 0.55; P < 0.01). Compared with Hereford calves, Angus calves exhibited greater serum but lower hair cortisol, greater PGEM, more lying bouts, and less healed wound scores at 3, 4, and 5 wk. Compared with heifers, steers exhibited lower PGEM, lower branding site and ocular IRT, higher MNT, and lower plasma meloxicam levels. Steers spent more time lying, took more lying bouts and had greater VAS pain, and more healed wound scores at 5 wk than heifers. Meloxicam administration at branding reduced branding and control site temperature differences and reduced lying bouts for the first 12 h. Breed and sex effects were observed across many biomarkers. Changes from baseline values for IRT, MNT, lying time, step count, VAS pain, and wound scoring all support that branding cattle is painful.

Hot-iron branding uses thermal injury to permanently identify cattle causing painful tissue damage. The primary objective was to examine the effects of oral meloxicam (MEL), compared with a control, administered at the time of hot-iron branding in Angus and Hereford steers and heifers. The secondary objectives were to investigate breed and sex effects on pain biomarkers. A total of 70 yearlings, consisting of 35 heifers and 35 steers (Angus, Hereford, or Angus × Hereford), were enrolled. Animals were assigned to receive MEL or a placebo. Changes from baseline values for infrared thermography (IRT), mechanical nociceptive threshold, lying time, step count, visual analog scale score, and wound scoring all support that hot-iron branding cattle is painful and investigation into analgesic strategies is needed. MEL administration reduced IRT differences from the branding and control site and reduced lying bouts. Breed and sex effects were observed across a wide range of biomarkers and should be considered in future pain studies. The practicality of administering a nonsteroidal anti-inflammatory drug once at the time of branding is attractive. However, a multimodal approach using a combination of analgesics or longer acting analgesic option warrants further investigation to alleviate pain and discomfort caused by hot-iron branding.