Evaluation of Fecal Egg Count Tests for Effective Control of Equine Intestinal Strongyles.

The American Association of Equine Practitioners strongly advocates evidence-based intestinal strongyle control in horses. It recommends targeted treatment of all heavy egg shedders (>500 eggs per gram (EPG) of feces), while the low shedders (0-200 EPG) are left untreated. As 50-75% of adult horses in a herd are low shedders, preventing them from unnecessary anthelmintic exposure is critical for tackling resistance. There are various fecal egg count (FEC) techniques with many modifications and variations in use, but none is identified as a gold standard. The hypothesis of the study was that the diagnostic performance of 12 commonly used quantitation methodologies (three techniques with four variants) differs. In this regard, method comparison studies were performed using polystyrene beads as proxy for intestinal strongyle eggs. Mini-FLOTAC-based variants had the lowest coefficient of variation (CV%) in bead recovery, whereas McMaster variants had the highest. All four variants of Mini-FLOTAC and the NaNO3 1.33 specific gravity variant of modified Wisconsin followed a linear fit with R2 > 0.95. In contrast, the bead standard replicates for modified McMaster variants dispersed from the regression curve, causing a lower R2. The Mini-FLOTAC method seems less influenced by the choice of floatation solution and has better repeatability parameters and linearity for bead standard recovery. For FEC tests with high R2 (>0.95) but that underestimated the true bead count, a correction factor (CF) was determined to estimate the true count. Finally, the validity of CF was analyzed for 5 tests with R2 > 0.95 to accurately quantify intestinal strongyle eggs from 40 different horses. Overall, this study identified FEC methodologies with the highest diagnostic performance. The limitations in standardizing routine FEC tests are highlighted, and the importance of equalization of FEC results is emphasized for promoting uniformity in the implementation of parasite control guidelines.

[Outbreak of Staphylococcus aureus mastitis in a dairy herd: A case report]. / Gehäuftes Auftreten von Staphylococcus-aureus-Mastitiden in einer Milchviehherde: Ein Fallbericht.

The report describes an outbreak of Staphylococcus aureus mastitis in a dairy herd. The risk assessment included the analysis of data from milk recordings, bacteriological milk cultures, clinical mastitis records and influence of infected status on the culling risk of affected animals, as well as an evaluation of the milking routine. The milking routine and the treatment protocol of animals with Staphylococcus aureus infection were identified as possible risk factors. The implemented measures included changes to the milking routine, a different treatment protocol for infected animals, and culling and segregation of infected animals to reduce overall prevalence.

Hyperketonemia: A Marker of Disease, a Sign of a High-Producing Dairy Cow, or Both?

This review covers the history and nomenclature of ketosis, the source and use of ketones in transition cows, and the controversial role of hyperketonemia's association with health and production outcomes in dairy cows. With the goal of assisting veterinarians with on-farm diagnostic and treatment methods, the authors present current and evolving means of direct and indirect hyperketonemia detection as well as a summary of treatment modalities and their efficacy. They encourage veterinarians to include hyperketonemia testing as part of their routine physical examinations and contemplate day in milk at hyperketonemia diagnosis when designing treatment and management strategies.

Metabolic Factors at the Crossroads of Periparturient Immunity and Inflammation.

Periparturient cows have the highest risk for disease and culling in the adult dairy herd. This risk is compounded by the multiple physiological changes of metabolism and immune function occurring around calving that alter the cow's inflammatory response. In this article, the authors summarize the current knowledge on immunometabolism in the periparturient cow, discussing major changes in immune and metabolic function around parturition that will facilitate the assessment of periparturient cow management programs.

The effect of heat treatment on colostral and newborn calf redox status and oxylipid biomarkers.

Newborn calves experience altered redox balance upon transition to extrauterine life. In addition to its nutritional value, colostrum is rich in bioactive factors, including pro- and antioxidants. The objective was to investigate differences in pro- and antioxidants as well as oxidative markers in raw and heat-treated (HT) colostrum and in the blood of calves fed either raw or HT colostrum. Eleven colostrum samples (≥8 L) of Holstein cows were each divided into a raw or HT (60°C, 60 min) portion. Both treatments were stored for <24 h at 4°C and tube-fed in a randomized-paired design at 8.5% of body weight to 22 newborn female Holstein calves within 1 h after birth. Colostrum samples were obtained before feeding, and calf blood samples were taken immediately before feeding (0 h) and at 4, 8, and 24 h after feeding. All samples were analyzed for reactive oxygen and nitrogen species (RONS) and antioxidant potential (AOP), from which the oxidant status index (OSi) was calculated. In 0-, 4-, and 8-h plasma samples, targeted fatty acids (FA) were analyzed using liquid chromatography-mass spectrometry, and oxylipids and isoprostanes (IsoP) using liquid chromatography-tandem mass spectrometry. Results for RONS, AOP, and OSi were analyzed by mixed-effects ANOVA or mixed-effects repeated-measures ANOVA, for colostrum and calf blood samples, respectively, whereas FA, oxylipid, and IsoP were analyzed using false discovery rate-adjusted analysis of paired data. Compared with control, HT colostrum showed lower RONS [least squares means (LSM) 189, 95% confidence interval (95% CI): 159-219 vs. 262, 95% CI: 232-292) relative fluorescence units] and OSi (7.2, 95% CI: 6.0-8.3 vs. 10.0, 95% CI: 8.9-11.1), but AOP remained unchanged (26.7, 95% CI: 24.4-29.0 vs. 26.4, 95% CI: 24.1-28.7 Trolox equivalents/µL). Changes in colostrum oxidative markers due to heat treatment were minor. No changes in RONS, AOP, OSi, or oxidative markers were detected in calf plasma. In both groups of calves, plasma RONS activity declined considerably at all postfeeding time points compared with precolostral values, and AOP reached its maximum 8 to 24 h after feeding. Generally, oxylipid and IsoP plasma abundance reached nadirs at 8 h post-colostrum in both groups. Overall, effects due to heat treatment on redox balance of colostrum and newborn calves and on oxidative biomarkers were minimal. In this study, heat treatment of colostrum reduced RONS activity but did not lead to detectable changes in calf oxidative status overall. This indicates that there were only minor changes in colostral bioactive components that could alter newborn redox balance and markers of oxidative damage.

Development of a bovine continuing education program for early-career veterinarians to address clinical service shortage.

There is currently a shortage of production animal veterinarians worldwide. Access to adequate continuing education (CE) increases retention of health-care workers in rural areas. However, little information is available about the preferences of bovine veterinarians in their first years after graduation. Our objective was to design a readily available CE program tailored for recent veterinary graduates working in bovine medicine. For this, we conducted a cross-sectional survey to identify early-career bovine veterinarian needs and their preferences to access CE conducted while practicing full time. Subsequently, we conducted a second survey to triangulate their responses with the opinions of experienced veterinarians, where the experienced veterinarians ranked the relevance of the CE topics that resulted from the early-career veterinarian survey. We received a total of 132 valid responses from US veterinarians in their first 5 years after graduation, and 32 responses associated with bovine veterinarians with 10 or more years of experience. Our results showed that a combination of distance education and workshops to practice hands-on skills was the preferred method for accessing CE among early-career veterinarians. Furthermore, recent graduates were willing to commit 1 to 2 h/wk to distance education. From the list of 20 topics identified by early-career veterinarians, the experienced veterinarians ranked "calf/heifer management" and "immunology and vaccinology" as the most relevant ones for practice. With the information gathered from these surveys, we designed, implemented, and piloted a 180-h CE program on bovine health management that is delivered over 2 yr through asynchronous distance education and annual hands-on workshops. Participant feedback has been very positive. Overall, the survey results will serve to develop CE programs targeted to bovine veterinarians in their first years of practice. Future research is needed to evaluate this program's success in retaining the bovine veterinary workforce within the United States, particularly in rural underserved areas.

Case-control exercise challenge study on the pathogenesis of high serum gamma-glutamyl transferase activity in racehorses.

BACKGROUND: High serum γ-glutamyl-transferase (GGT) activity syndrome in racehorses has been associated with maladaption to exercise. Investigation of affected horses before and immediately after standard exercise may provide critical insight into the syndrome's pathophysiology. OBJECTIVES: To investigate blood biomarker changes in actively competing racehorses with high GGT activity associated with an exercise challenge. STUDY DESIGN: Case-control study. METHODS: High GGT case (age: 2-3 years) and normal GGT control (age: 2-7 years) pairs (3 Thoroughbred, 4 Standardbred pairs) at least 3 months into their training/racing season were included. Horses with a recent history of high GGT activity (≥50 IU/L) without additional biochemical evidence of liver disease were identified by veterinarians. Horses were tested again in the week prior to a planned exercise challenge to confirm persistent increases in GGT activity. Controls from the same stable with similar training/racing intensity and serum GGT activity ≤36 IU/L were matched with each case. Blood samples were obtained immediately before, 15 and 120 min after exercise. Pre-exercise serum samples were analysed for baseline select serum chemistries, selenium and vitamin E concentrations. Cortisol concentration and markers of oxidative status were measured in serum or plasma for all time points. Individual serum bile acid and coenzyme Q10 concentrations, plasma lipid mediator (fatty acids, oxylipids, isoprostanes) concentrations and targeted metabolomics analyses were performed using liquid chromatography-mass spectrometry. Serum viral PCR for equine hepaci- and parvovirus was performed in each animal. RESULTS: Cases had higher baseline concentrations of total glutathione, taurocholic acid, cortisol and cholesterol concentrations and higher or lower concentrations of specific oxylipid and isoprostane mediators, but there were no case-dependent changes after exercise. MAIN LIMITATIONS: Small sample size. CONCLUSIONS: Results indicated that glutathione metabolism was altered in high GGT horses. Enhanced glutathione recycling and mild cholestasis are possible explanations for the observed differences.

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant.

The quantification of cytokines can improve our understanding of immune response and inflammation dynamics in dairy cows. Bead-based assays provide a sensitive, high-throughput platform, allowing for simultaneous quantification of multiple cytokines within a wide linear detection range. Our objective was to develop a multiplex bead-based assay using monoclonal antibodies for simultaneous quantification of bovine tumor necrosis factor (TNF)-α, IL-10, and IFN-γ in plasma and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant cytokine standards produced in mammalian cells were used to determine the lower limit of detection and the linear detection range for each cytokine. The lower limit of detection was 110 pg/mL for IL-10, 95 pg/mL for TNF-α, and 20 pg/mL for IFN-γ. The linear quantification range was 110 to 241,000 pg/mL for IL-10, 95 to 620,000 pg/mL for TNF-α, and 20 to 130,000 pg/mL for IFN-γ. All 3 monoclonal capture and detection antibodies were specific for their respective cytokine analyte when using the recombinant IL-10, TNF-α, and IFN-γ standards. Intraassay and interassay coefficients of variation (CV) were <10% and <12%, respectively, for all analytes and samples matrices. Next, concentrations of native cytokines were determined in PBMC culture supernatants (n = 4) and in plasma from whole-blood samples (n = 6) with or without stimulation with Escherichia coli lipopolysaccharide or a mix of phorbol myristate acetate (PMA) and ionomycin. Peak concentrations of all 3 cytokines were secreted from PBMC after PMA/ionomycin stimulation (TNF-α, 8 h, range: 39,266-506,422 pg/mL; IL-10, 18 h, range: 15,770-63,415 pg/mL; IFN-γ 18 h, range: 189,977-492,659 pg/mL). In contrast, the highest concentrations in plasma from whole-blood stimulation were observed for IL-10 and TNF-α after LPS stimulation (TNF-α, 4 h, range: 1,764-13,460 pg/mL; IL-10, 24 h, range: 2,401-6,371 pg/mL), whereas PMA and ionomycin induced the highest secretion of IFN-γ (18 h, range: 53-20,215 pg/mL). In conclusion, the multiplex assay can quantify native IL-10, TNF-α, and IFN-γ across a broad concentration range in bovine plasma and cell culture supernatant, thereby providing a novel tool to evaluate inflammatory profiles in cattle and especially in dairy cows with inflammatory conditions. The existing multiplex assay can be expanded in the future by adding bead assays for additional bovine cytokines.

Inhibition of mTOR in bovine monocyte derived macrophages and dendritic cells provides a potential mechanism for postpartum immune dysfunction in dairy cows.

Dairy cattle experience a profound nutrient deficit postpartum that is associated with immune dysfunction characterized by heightened inflammation and reduced pathogen clearance. The activation of the central nutrient-sensing mTOR pathway is comparatively reduced in leukocytes of early postpartum dairy cows during this time of most pronounced nutrient deficit. We assessed the effect of pharmacological mTOR inhibition (Torin-1, rapamycin) on differentiation of monocyte derived classically (M1) and alternatively (M2) activated macrophages (MPh) and dendritic cells (moDC) from 12 adult dairy cows. Treatment with mTOR inhibitors generated M1 MPh with increased oxidative burst and expression of IL12 subunits but decreased phagocytosis and expression of IL1B, IL6, and IL10. In M2 MPh, treatment inhibited expression of regulatory features (CD163, ARG2, IL10) skewing the cells toward an M1-like phenotype. In moDC, mTOR inhibition increased expression of pro-inflammatory cytokines (IL12A, IL12B, IL1B, IL6) and surface CD80. In co-culture with mixed lymphocytes, mTOR-inhibited moDC exhibited a cytokine profile favoring a Th1 response with increased TNF and IFNG production and decreased IL10 concentrations. We conclude that mTOR inhibition in vitro promoted differentiation of inflammatory macrophages with reduced regulatory features and generation of Th1-favoring dendritic cells. These mechanisms could contribute to immune dysregulation in postpartum dairy cows.

Effects of dietary branched-chain amino acid supplementation on serum and milk metabolome profiles in dairy cows during early lactation.

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.

Pharmacological inhibition of the mTOR pathway alters phenotype and cytokine expression in bovine monocyte-derived dendritic cells.

Epidemiological studies have long demonstrated the association of nutrient status and immune dysfunction in dairy cows. Postpartum dairy cows experiencing a nutrient deficit show a propensity for increased inflammatory response, decreased pathogen clearance, and increased incidence of infectious disease. Studies in cows and other species show that the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway could be one potential causal pathway connecting the deficit in nutrient availability and the heightened inflammatory response. Our objective was to investigate the effects of pharmacological mTOR pathway inhibition on phenotype and cytokine expression of bovine monocyte derived dendritic cells (moDC). We differentiated CD14+ monocytes from dairy cows (n = 14) into moDC in the presence or absence of first- or second-generation mTOR inhibitor rapamycin and PP242 (both 100 nM), respectively. On day seven cells were matured with E. coli lipopolysaccharide (LPS, 100 ng/mL) or left unstimulated to represent naïve moDC. Surface expression of CD14, CD40, CD80, and MHCII was measured via flow cytometry. We measured mRNA expression of IL10, IL12A, IL12B, and TNFα by rt-qPCR, and protein concentrations of IL-10 and TFN-α in cell culture supernatants with a bead-based multiplex assay. Cultures from ten cows successfully developed the moDC phenotype in culture without inhibitors, defined as increased surface expression of CD40, CD80, and MHCII compared with naïve moDC. Only data from these cows were considered for the results on effects of mTOR inhibitors. In naïve and mature moDC mTOR inhibition increased MHCII expression compared to controls. In mature moDC, in addition to MHCII, CD80 expression was increased compared with untreated LPS-stimulated controls. Expression of IL12A mRNA was upregulated in mature, mTOR inhibited moDC compared with untreated controls. In cell culture supernatants mTOR inhibition reduced IL-10 and increased TNF-α concentrations in naïve and mature moDCs compared with untreated controls. Overall rapamycin had a more consistent effect on altering phenotype and cytokine expression of moDC than PP242. In summary we observed an increased expression of co-stimulatory molecules and antigen presentation potential in mature moDC differentiated under mTOR inhibition, and a cytokine pattern that would potentially favor a Th1 type response. This study provides novel data indicating a role for mTOR signaling in bovine moDC phenotype and mediator profile. This proof-of-concept study demonstrates the role of the mTOR pathway in shaping the bovine immune response and may help to provide mechanistic insight and opportunities for modulation of the immune response during the nutrient deficit of early lactation.

Investigating the pathogenesis of high-serum gamma-glutamyl transferase activity in Thoroughbred racehorses: A series of case-control studies.

BACKGROUND: High-serum γ-Glutamyl Transferase (GGT) activity has been associated with and thought to be a marker of maladaptation to training and possibly poor performance in racehorses, but the cause is unknown. OBJECTIVES: To investigate possible metabolic and infectious causes for the high GGT syndrome. STUDY DESIGN: Pilot case-control study and nested case-control study. METHODS: The case-control study in 2017 included 16 horses (8 cases and 8 controls with median [range] serum GGT 82 [74-148] and 22 [19-28] IU/L, respectively) from the same stable. In 2018, similar testing was performed in a nested case-control study that identified 27 case (serum GGT 50 ≥ IU/L)-control pairs from three stables for further testing. Serum liver chemistries, selenium measurements, viral PCR and metabolomics were performed. RESULTS: No differences were found in frequency of detection of viral RNA/DNA or copy numbers for equine hepacivirus (EqHV) and parvovirus-hepatitis (EqPV-H) between cases and controls. Mild increases in hepatocellular injury and cholestatic markers in case vs control horses suggested a degree of liver disease in a subset of cases. Metabolomic and individual bile acid testing showed differences in cases compared with controls, including increased abundance of pyroglutamic acid and taurine-conjugated bile acids, and reduced abundance of Vitamin B6. Selenium concentrations, although within or above the reference intervals, were also lower in case horses in both studies. MAIN LIMITATIONS: Observational study design did not allow us to make causal inferences. CONCLUSIONS: We conclude that high GGT syndrome is likely a complex metabolic disorder and that viral hepatitis was not identified as a cause for this syndrome in this cohort of racehorses. Our results support a contribution of oxidative stress and cholestasis in its pathophysiology.

Ambidirectional cohort study on the agreement of ultrasonography and surgery in the identification of parathyroid pathology, and predictors of postoperative hypocalcemia in 47 dogs undergoing parathyroidectomy due to primary hyperparathyroidism.

OBJECTIVE: To investigate (1a) agreement of ultrasonographic, surgical, and histopathologic findings in the diagnosis of a neoplastic etiology underlying primary hyperparathyroidism (PHPT), (1b) the ability of ultrasonographically determined parathyroid gland size to distinguish between malignant (carcinoma) and non-malignant (hyperplasia, adenoma) pathology, and (2) variables associated with postoperative hypocalcemia in dogs undergoing surgical treatment of PHPT. STUDY DESIGN: Ambidirectional cohort study. ANIMALS: Forty-seven client owned dogs with PHPT (34 retrospective; 13 prospective). METHODS: Data were extracted from medical records. Method agreements were explored using Cohen's Kappa statistic. A receiver operating characteristic curve (ROC) was used to determine a cut-off separating parathyroid pathologies. Univariable and multivariable models assessed associations between postoperative hypocalcemia and potential risk factors. RESULTS: Agreement of ultrasound and surgery for number and side of affected glands was 31/47 (65.9%) and 34/47 (72.3%), respectively. In 37/47 (78.7%) cases, parathyroid tissue was correctly assessed as pathologic by the surgeon. An ultrasonographic cut-off of ≥8.0 mm (ROC AUC = 0.82) best distinguished malignant from benign pathologies. Dogs with a preoperative serum ionized calcium (iCa) concentration ≥1.75 mEq/L had 7.5 times greater odds of becoming hypocalcemic postoperatively. CONCLUSION: A fair agreement existed between ultrasonographic and surgical findings in dogs with PHPT. A parathyroid mass ≥8.0 mm on ultrasonographic examination was suggestive of malignancy, while dogs with a preoperative serum iCa concentration ≥1.75 mEq/L were at increased risk for postoperative hypocalcemia in this study. CLINICAL SIGNIFICANCE: This study supports the use of bilateral cervical surgical exploration to identify abnormal parathyroid glands for the treatment of PHPT.

Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α.

The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

Agreement of stall-side and laboratory major crossmatch tests with the reference standard method in horses.

BACKGROUND: Crossmatching is used to prevent life-threatening transfusion reactions in horses. Laboratory methods are laborious and technically challenging, which is impractical during emergencies. HYPOTHESIS/OBJECTIVES: Evaluate agreement between a stall-side crossmatch kit (KIT) and a laboratory method (LAB) in horses with known and unknown blood types. ANIMALS: Twenty-four blood-typed and alloantibody-screened healthy adult horses (Aim 1) and 156 adult horses of unknown blood type (Aim 2). METHODS: Prospective, blinded study. Expected positive (n = 35) and negative (n = 36) crossmatches among 24 antibody and blood-typed horses were used to determine sensitivity and specificity of KIT and LAB against the reference method. Agreement in 156 untyped horses was evaluated by reciprocal crossmatch (n = 156). RESULTS: Sensitivity (95% confidence interval [CI]) for LAB and KIT compared with expected reactions was 77.1% (59.9%-90.0%) and 91.4% (77.0%-98.2%), and specificity 77.8% (60.9%-89.9%) and 73.5% (55.6%-87.1%), respectively. The KIT was 100% sensitive for Aa reactions; LAB was 100% sensitive for Qab; and both were 100% sensitive for Ca. Cohen's κ agreement for LAB and KIT with expected positive and negative reactions (n = 71) was moderate (0.55 [0.36-0.74]) and substantial (0.65 [0.47-0.82]), respectively. Agreement was fair comparing LAB with KIT in Aim 1 (0.30 [0.08-0.52]) and in untyped horses in Aim 2 (0.26 [0.11-0.41]). CONCLUSIONS AND CLINICAL IMPORTANCE: Agreement between KIT and LAB with expected reactions was blood type dependent. Performance of both methods depends on blood type prevalence.

Blood ß-hydroxybutyrate concentrations and early lactation management strategies on pasture-based dairy farms in Colombia.

The increasing global demand for food requires sustainable solutions to close the gap in agricultural yield between industrialized and non-industrialized countries. Our objectives in this cross-sectional study were to: 1) characterize farm populations, milk yield, and early lactation management strategies of dairy cows in three different regions of Colombia, and 2) determine the association of these management strategies with blood ß-hydroxybutyrate (BHB) concentrations in the first 42 days in milk (DIM). Dairy herds (n = 56) in the Antioquia, Caldas, and Cundinamarca regions of Colombia were visited once from May through July 2018. A survey was administered to farm owners to collect demographic, management, and herd nutrition information. Blood samples from dairy cows (n = 880) between calving and 42 DIM were used to measure blood BHB concentration. Associations between management and nutritional strategies and blood BHB concentration were examined using mixed models. Prevalence of hyperketonemia was calculated as the number of samples with BHB concentration ≥1.2 mmol/L divided by the total number of samples. The estimated diet composition for early lactation dairy cows was 65.5% pasture and 31.8% commercial concentrates. The farm median milk yield, protein concentration, and fat concentration were 21.0 kg (range = 13.1-36 kg), 3.2% (range = 2.7-4.1%), and 3.5% (range = 3.0-4.1%), respectively. Milk yield least squares means (95% confidence interval; CI) differed by region: 21.7 (20.3, 23.2), 18.5 (17.0, 20.2), and 20.3 (18.5, 22.4) kg in Antioquia, Caldas, and Cundinamarca, respectively. Median blood BHB concentration was 0.5 and ranged from 0.1-4.4 mmol/L; blood BHB concentration was not different among the three regions. Pasture fertilization, increased parity, and BCS were associated with changes in blood BHB concentration. The overall prevalence of hyperketonemia was 4.5%. Geographical region affected the prevalence of hyperketonemia at 2.5%, 4.0%, and 10.2% in Antioquia, Caldas, and Cundinamarca, respectively. Mean stocking density (95% CI) was greater in Cundinamarca than Antioquia or Caldas at 3.3 (2.2, 5.0), 2.8 (2.1, 3.9) and 1.7 (1.2, 2.6) animals per ha, respectively, and was associated with hyperketonemia prevalence. Farms that abruptly stop milking cows at dry-off had 80% of the hyperketonemia events in the study. Pasture-based dairies in Colombia had lower blood BHB concentrations and estimated milk yield compared with confined production systems in temperate zones. However, geographical region, stocking density, and abrupt cessation of milking at dry-off were associated with prevalence of hyperketonemia in pasture-based dairies.

The effect of ex vivo lipopolysaccharide stimulation and nutrient availability on transition cow innate immune cell AKT/mTOR pathway responsiveness.

Postpartum dairy cows experience a heightened inflammatory state coinciding with the time of greatest nutrient deficit. Nutrient availability is sensed on the cellular level by nutrient sensing kinases, such as the PI3K/AKT/mTOR (mTOR) pathway, a key orchestrator of immune cell activation and inflammatory balance. Our objective was to determine the responsiveness of this pathway to inflammatory stimulation with and without nutrient supplementation ex vivo. Blood samples were collected from Holstein cows (n = 14) at -42, -14, 7, 21, and 42 d relative to calving. Control samples and samples pretreated with a mixture of amino acids, glucose, and insulin (AAM) were stimulated with 100 ng/mL E. coli lipopolysaccharide (LPS; LPS, AAMLPS) or left unstimulated (control, AAM). After 1 h, ratios of mean fluorescence intensity for phosphorylated to total protein of AKT and mTORC1 substrates S6RP and 4EBP1 were analyzed in polymorphonuclear cells (PMN), and monocytes by flow cytometry. A separate aliquot was stimulated with LPS for 2 h and relative mRNA abundance of IL10, IL12A, IL12B, and TNFA in whole blood leukocytes from 10 cows was measured by reverse-transcription quantitative PCR. Repeated measures ANOVA was performed with fixed effects of time, treatment, and their interaction. Cells had different ratios of pathway proteins with PMN having the highest phosphorylation of AKT, S6RP, and 4EBP1. Stimulation with LPS consistently activated mTOR signaling in PMN regardless of nutrient supplementation except for postpartum 4EBP1, which increased in response to nutrients alone. In monocytes, AKT baseline phosphorylation was lower and activation could not be induced by either treatment, whereas activation of 4EBP1 responded to nutrient supplementation. Treatment with LPS increased phosphorylation of S6RP in both innate immune cell types. Nutrient supplementation increased baseline IL10 expression and decreased baseline as well as LPS-induced IL12B and TNFA expression. We conclude that the mTOR pathway in bovine innate immune cells can be differentially activated in response to inflammatory stimulation and nutrient supplementation in monocytes versus PMN. Effects of nutrient supplementation on cytokine mRNA abundance are likely specific to immune cell type.

Longitudinal Phenotypes Improve Genotype Association for Hyperketonemia in Dairy Cattle.

The objective of our study was to identify genomic regions associated with varying concentrations of non-esterified fatty acid (NEFA), ß-hydroxybutyrate (BHB), and the development of hyperketonemia (HYK) in longitudinally sampled Holstein dairy cows. Our study population consisted of 147 multiparous cows intensively characterized by serial NEFA and BHB concentrations. To identify individuals with contrasting combinations in longitudinal BHB and NEFA concentrations, phenotypes were established using incremental area under the curve (AUC) and categorized as follows: Group (1) high NEFA and high BHB, group (2) low NEFA and high BHB), group (3) low NEFA and low BHB, and group (4) high NEFA and low BHB. Cows were genotyped on the Illumina Bovine High-density (777K) beadchip. Genome-wide association studies using mixed linear models with the least-related animals were performed to establish a genetic association with HYK, BHB-AUC, NEFA-AUC, and the comparisons of the 4 AUC phenotypic groups using Golden Helix software. Nine single-nucleotide polymorphisms were associated with high longitudinal concentrations of BHB and further investigated. Five candidate genes related to energy metabolism and homeostasis were identified. These results provide biological insight and help identify susceptible animals thus improving genetic selection criteria thereby decreasing the incidence of HYK.

Internet Survey of Participant Demographics and Risk Factors for Injury in Flyball Dogs.

Background: Injury risk in canine sprinting sports, such as greyhound racing and agility, have been previously documented through various surveys. Flyball, another sprinting canine sport with similar athletic requirements to agility, has yet to be assessed for factors associated with injury. The aim of this study was to determine injury prevalence and assess for risk factors for injury in flyball dogs. Methods: Survey data from 375 flyball participants was collected and analyzed. Data collected included patient-specific variables, equipment use, training/competition practices, and injury occurrence and localization. Univariate analysis was utilized for all variables of interest, followed by backwards nominal logistic regression to identify variables associated with increased risk of injury, with a p < 0.05 defined as significant. Results: Thirty-nine percent of dogs incurred at least one injury with 172 injuries reported. Injuries to the limbs were common (30.8% affecting forelimbs, 25.6% affecting hindlimbs), with the paw or nail the most frequently reported injured area (19.2%). Only protective wrap use and age were significantly associated with injury in the final nominal regression model (p < 0.01). A biphasic injury rate with more injuries in younger dogs was observed, and injuries peaked by 6 years of age. Use of carpal wraps was positively associated with increased injury risk. Conclusions: These findings suggest an association between younger dogs and greater risk of injury, as well as identify a need to further investigate the utilization of wraps and potential association between injury risk among flyball participants.