Molecular evidence sterile tissue damage during pathogenesis of pododermatitis aseptica hemorrhagica circumscripta is associated with disturbed epidermal-dermal homeostasis.

Podermatitis aseptica hemorrhagica circumscripta is associated with metalloproteinase 2 weakening of distal phalangeal suspensory structures and sinkage of the distal phalanx in the claw capsule. Pressure from the tuberculum flexorium on the sole epidermis and dermis produces hemorrhagic tissue injury and defective horn production appearing as yellow-red, softened claw horn in region 4 of the sole. A model of the MAPK/ERK signal cascade orchestrating epidermal-dermal homeostasis was employed to determine if sterile inflammatory responses are linked to disturbed signal transduction for epidermal homeostasis in sole epidermis and dermis. The objective was to assess shifts in target genes of inflammation, up- and downstream MAPK/ERK signal elements, and targeted genes supporting epidermal proliferation and differentiation. Sole epidermis and dermis was removed from lateral claws bearing lesions of podermatitis aseptica hemorrhagica circumscripta, medial claws from the same limb and lateral claws from completely normal limbs of multiparous, lactating Holstein cows. The abundance levels of targeted transcripts were evaluated by real-time QPCR. Lesion effects were assessed by ANOVA, and mean comparisons were performed with t-tests to assess variations between mean expression in ulcer-bearing or medial claw dermis and epidermis and completely normal lateral claw dermis and epidermis or between ulcer-bearing dermis and epidermis and medial claw dermis and epidermis. The lesions were sterile and showed losses across multiple growth factors, their receptors, several downstream AP1 transcription components, CMYC, multiple cell cycle and terminal differentiation elements conducted by MAPK/ERK signals and ß 4, α 6 and collagen 17A hemidesmosome components. These losses coincided with increased cytokeratin 6, ß 1 integrin, proinflammatory metalloproteinases 2 and 9, IL1B and physiologic inhibitors of IL1B, the decoy receptor and receptor antagonist. Medial claw epidermis and dermis from limbs with lateral claws bearing podermatitis aseptica hemorrhagica circumscripta showed reductions in upstream MAPK/ERK signal elements and downstream targets that paralleled those in hemorrhagic lesions. Inhibitors of IL1B increased in the absence of real increases in inflammatory targets in the medial claw dermis and epidermis. Losses across multiple signal path elements and downstream targets were associated with negative effects on targeted transcripts supporting claw horn production and wound repair across lesion-bearing lateral claws and lesion-free medial claw dermis and epidermis. It was unclear if the sterile inflammation was causative or a consequence of these perturbations.

Transcriptional responses consistent with perturbation in dermo-epidermal homeostasis in septic sole ulceration.

The aim of this study was to evaluate transcriptional changes in sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis was removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis was obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time QPCR and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of pro-inflammatory transcripts interleukin 1 α (IL1A), interleukin1 ß (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), and caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA) and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.

Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows.

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the ß2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by ß2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3+CD4+ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3+ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ+ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3+CD8+ cytotoxic T cells and CD3+CD11c+ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.

Bovine coronary region keratinocyte colony formation is supported by epidermal-dermal interactions.

Delineating the factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronary region keratinocytes and dermal fibroblasts, determine the colony-forming capacity of epidermal keratinocytes in the coronary region, and characterize transcriptional changes in specific cytokine, growth factor, and receptor genes during colony formation in coculture. Fibroblasts and keratinocytes from the coronary region of the lateral, hind limb claw were collected, and 5.0 x 10(3) and 7.5 x 10(3) keratinocytes were cultured in the presence or absence of fibroblast monolayers, respectively. The 2 densities of keratinocytes formed 144 +/- 15.8 and 183 +/- 26.9 colonies, respectively, in the presence of dermal fibroblasts, whereas no colonies developed in the absence of dermal fibroblasts. Keratinocytes with the ability to show colony formation comprised 1.09% +/- 0.16 to 1.77% +/- 0.28 of the keratinocyte population isolated from the coronary region. Keratinocyte-fibroblast cocultures developed a time-dependent increased expression of several growth factors, cytokines, and receptors. These findings demonstrated that keratinocytes from the bovine coronary region formed colonies in vitro and that colony formation occurred with an absolute dependence on dermal fibroblasts. Colony growth was associated with increased transcriptional expression of cytokine, growth factor, and receptor expression known to drive keratinocyte colony formation in other species. The results indicate that horn-producing keratinocytes must interact with dermal fibroblasts during normal tissue homeostasis in the bovine claw.

Enhancing the prediction accuracy of bovine lameness models through transformations of limb movement variables.

The issue of modeling bovine lameness was explored by testing the hypothesis that B-spline transformation of limb movement variables (LMV) employed in predictive models improved model accuracy. The objectives were to determine the effect of number of B-spline knots and the degree of the underlying polynomial approximation (degree of freedom) on model accuracy. Knot number used in B-spline transformation improved model accuracy by improving model specificity and to a lesser extent model sensitivity. Degree of polynomial approximation had no effect on model predictive accuracy from the data set of 261 cows. Model stability, defined as changes in predictive accuracy associated with the superimposition of perturbations (0.5 and 1.0%) in LMV on the measured data, was explored. Model specificity and to a lesser degree, sensitivity, increased with increased knot number across data set perturbations. Specificity and sensitivity increased by 43 and 11%, respectively, when knot number increased from 0 to 7 for a perturbation level of 0.5%. When the perturbation level was 1%, the corresponding increases in specificity and sensitivity were 32 and 4%, respectively. Nevertheless, different levels of LMV perturbation varied the optimal knot number associated with highest model accuracy. The optimal knot number for 0.5% perturbation was 8, whereas for 1% perturbation the optimal knot number was 7. The B-spline transformation improved specificity and sensitivity of predictive models for lameness, provided the appropriate number of knots was selected.

Age, segment, and horn disease affect expression of cytokines, growth factors, and receptors in the epidermis and dermis of the bovine claw.

The aim of this study was to examine changes in RNA expression for growth factors, cytokines, and receptors in epidermal-dermal tissues of the bovine claw relative to host age, claw segment, and disease state of the horn. Epidermal-dermal tissues were collected from the coronary, wall, sole, and bulb segments of 8- to 9-mo-old Holstein fetuses, normal adult cows, and adult cows with sole ulceration. Anatomic and pathologic characteristics were determined in tissues stained with eosin and hematoxylin, and RNA expression levels were evaluated using real-time, quantitative PCR. In normal tissues, certain RNA expression levels were clearly affected by host age: 290.0-, 610.0-, 53.4-, and 8.1-fold greater expression of granulocyte-macrophage colony stimulating factor was observed in fetal coronary, wall, sole, and bulb segment relative to adult tissues, respectively. A claw segment effect was also observed in that IL-1alpha expression was greater (1.59-fold) in the normal adult wall relative to the coronary segment, and IL-18 expression was greater (16.2-fold) in the normal adult sole compared with the coronary segment and 2.88 greater in the fetal sole relative to the bulb segment. Sole ulceration was associated with hemorrhage, thrombosis, inflammation, and striking increases in IL-1beta, IL-18, and inducible nitric oxide synthase, and with less dramatic, albeit measurable, changes in IL-1 type I receptor, IL-1 receptor antagonist, and tumor necrosis factor-alpha. Amidst striking increases in keratinocyte growth factor receptor (i.e., 21.0-fold, 10.4-fold, 0, and 21.6-fold in the coronary, wall, sole, and bulb segments, respectively), a concomitant decrease occurred in keratinocyte growth factor (i.e., 0.80-, 0.54-, 0.56-, and 0.72-fold, respectively). The results demonstrated changes in disease state and, to a lesser extent, claw segment and were accompanied by alterations in the RNA expression of several cytokines, growth factors, and receptors present in the normal claw.

Objective determination of claw pain and its relationship to limb locomotion score in dairy cattle.

We hypothesized that claw and foot pain could be objectively determined and have a strong effect on limb locomotion. Claw pain was measured using hoof testers equipped with a pressure gauge. Soft tissue pain was evaluated with an algometer. Pain was determined as the maximum pressure recorded at the time the limb was withdrawn following claw or soft tissue compression with the hoof tester or algometer. Locomotion scores and claw and soft tissue pain were determined on 263 Holstein cows from 2 commercial dairy farms. The frequency and the magnitude of pain had an effect on locomotion score in the ipsilateral limb for lateral, but not medial, claws. The magnitude of the lateral claw pain index for limbs with locomotion scores 1 to 5 was 0.95 +/- 0.01, 0.90 +/- 0.02, 0.67 +/- 0.04, 0.65 +/- 0.05, and 0.45 +/- 0.11, respectively. The magnitude of the medial claw pain index for limbs with locomotion scores 1 to 5 was 1.0 +/- 0.00, 0.99 +/- 0.01, 0.98 +/- 0.01, 1.0 +/- 0.00, and 1.0 +/- 0.00, respectively. The frequency of painful claws (n = 208) in limbs with locomotion scores 1, 2, and > or =3 was 0.529, 0.173, and 0.298, respectively. The frequency of painless claws (n = 318) in limbs with locomotion scores 1, 2, or > or =3 was 0.792, 0.088, and 0.120, respectively. The frequency of pain (27.1%) and total lesions (85.6%) was greater in lateral claws (n = 524) than that of pain (2.1%) and total lesions (14.4%) in medial claws (n = 524). Yet the magnitude of the pain index in sore claws was similar for medial (0.73 +/- 0.09) and lateral claws (0.64 +/- 0.02). The magnitude and frequency of claw pain in one hind limb was inconsistently and weakly affected by locomotion score or claw pain in the contralateral limb. The prevalence of unilateral (32.8%) and bilateral (23.3%) pain was similar and lower than the occurrence of bilaterally nonpainful claws (43.9%) in the study group. Painful claws (n = 78) occurred on sound limbs (n = 332) with a pain index (0.72 +/- 0.02) indicative of less pain than the pain index (0.61 +/- 0.02) of painful claws (n = 130) on lame limbs (n = 192). The results showed that lateral claw pain was related to ipsilateral limb locomotion score and subclinical pain was a relatively common occurrence. Objective measures of pain may provide a more reliable, continuous measure of clinical events used in modeling lameness.

Comparison of models to identify lame cows based on gait and lesion scores, and limb movement variables.

Bovine lameness results in pain and suffering in cattle and economic loss for producers. A system for automatically detecting lame cows was developed recently that measures vertical force components attributable to individual limbs. These measurements can be used to calculate a number of limb movement variables. The objective of this investigation was to explore whether gait scores, lesion scores, or combined gait and lesion scores were more effectively captured by a set of 5 limb movement variables. A set of 700 hind limb examinations was used to create gait-based, lesion-based, and combined (gait- and lesion-based) models. Logistic regression models were constructed using 1, 2, or 3 d of measurements. Resulting models were tested on cows not used in modeling. The accuracy of lesion-score models was superior to that of gait-score models; lesion-based models generated greater values of areas under the receiving operating characteristic curves (range 0.75 to 0.84) and lower mean-squared errors (0.13 to 0.16) compared with corresponding values for the gait-based models (0.63 to 0.73 and 0.26 to 0.31 for receiving operating characteristic and mean-squared errors, respectively). These results indicate that further model development and investigation could generate automated and objective methods of lameness detection in dairy cattle.

Surface receptors for IgG and complement on equine alveolar macrophages.

Isolated equine alveolar macrophages obtained by bronchopulmonary lavage of four live ponies demonstrated surface receptors for equine IgG, equine IgM, and complement-coated sheep red blood cells, but not equine IgM or complement-coated erythrocytes alone. In addition, demonstration of IgG receptors was found to depend on the level of erythrocyte sensitization and could not be demonstrated by red blood cell rosetting techniques at low levels of sensitization. Demonstration of receptors for equine complement by red cell rosetting techniques required the presence of both IgM antibody and serum derived (complement) components. This is the first such study of receptors on equine alveolar macrophages.

Chemiluminescence response of equine alveolar macrophages during stimulation with latex beads, or IgG-opsonized sheep red blood cells.

Isolated equine alveolar macrophages were shown to generate a luminol-dependent light response when challenged with a phagocytic stimulus. The chemiluminescent response was not detected with luminol prepared at 1.0 x 10(-5) or 1.0 x 10(-4) molar concentrations, but was readily quantitated when used at a 1.0 x 10(-3) molar concentration. Challenge of the alveolar macrophages with latex particles or with equine IgG-coated sheep red blood cells elicited the luminol-dependent light response, whereas unchallenged equine alveolar macrophages or those challenged with unopsonized erythrocytes failed to emit light above background levels. Latex-bead-challenged macrophages released 8.06 times the total amount of light as those equine alveolar macrophages challenged with equine IgG-opsonized erythrocytes. This study represents the first investigation on chemiluminescence and equine alveolar macrophages.

Serologic survey of Neospora caninum infection in a closed dairy cattle herd in Maryland: risk of serologic reactivity by production groups.

Prevalence of antibodies to Neospora caninum was determined in a cross-sectional consensus survey of 1029 bovines in a dairy herd with endemic Neospora-induced abortion. Sera were screened by indirect fluorescent antibody test (IFAT). The prevalence of N. caninum antibody in the IFAT was 17.9% in 107 neonates, 26.2% in 233 yearling heifers and steers, 39.07% in 218 mature heifers, and 26.9% in 465 milking cows. Serologic reactivity was associated with production grouping on the farm with the greatest risk of serologic reactivity appearing in the yearling and mature heifers. There was an increasing risk of serologic reactivity with increasing age only in the parity one and greater animals in the herd. Castrated males were at half the risk of similarly aged females of possessing antibodies to N. caninum. There was no clear relationship between the serologic status of dams and offspring.

Influence of estradiol on duration of anestrus and incidence of short estrous cycles in postpartum cows.

The influence of exposure to exogenous estradiol on the interval from parturition to first ovulation, luteinizing hormone (LH) secretion and luteal function was examined in cows. Cows were assigned at parturition to one of three treatments. Cows received either a 3.0 (1-E; n = 30) or .75 cm (1/4-E; n = 28) implant containing 17 beta-estradiol or served as untreated control animals (C; n = 33). Implants were administered within 2 days following parturition and removed on day 40 postpartum (day 0 = day of parturition). Single blood samples were collected twice weekly and analyzed for progesterone to determine length of postpartum anestrus and duration of the initial increase in progesterone. Sequential blood samples were collected on day 35 +/- .1 postpartum (15 min intervals for 18 hrs) from 5 cows in each treatment and analyzed for LH. Concentrations of estradiol were higher (P less than .01) in the 1-E (5.3 +/- .24) than in C (3.9 +/- .23) or 1/4 E (3.9 +/- .25) cows on day 35 postpartum. The interval from parturition to the first estrous cycle of normal duration was similar for cows in the C and 1-E treatment (53 +/- 2.4 and 56 +/- 2.4 days, respectively). Cows in the 1/4-E treatment had a longer (P less than .05) interval (68 +/- 2.5 days). Secretion of LH was similar among treatments on day 35 postpartum. The first normal luteal phase after parturition was preceded by a transient rise in progesterone in 81, 64 and 85% of the cows in the C, 1-E and 1/4-E treatments, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cloprostenol sodium and clenbuterol HCl on reproductive performance in postpartum anestrous cows.

Two experiments were conducted to study effects of cloprostenol sodium (cloprostenol) and clenbuterol HCl (clenbuterol) during postpartum anestrus on subsequent reproductive performance in cows. In Experiment I, 96 cows received either 0.5 mg cloprostenol (PGF, n = 25), 364 mg clenbuterol (CLEN, n = 24), 0.5 mg cloprostenol and 364 mg clenbuterol (CLEN+PGF, n = 21) or no treatment (Control, n = 26) on Day 20 post partum. Treatments failed to influence postpartum interval, pregnancy rate or the incidence of short estrous cycles preceding the first normal estrous cycle. In Experiment II, anestrous cows were administered cloprostenol (0.5 mg) on either Day 20 (PGF20, n = 27) or Day 35 post partum (PGF35, n = 25), or served as untreated controls (Control, n = 26). Neither postpartum interval nor pregnancy rate were affected by cloprostenol treatment. In conclusion, treatment of postpartum cows with PGF did not alter the resumption of normal estrous cycles following parturition.

Oxidative metabolism of the bovine alveolar macrophage.

Oxidative respiratory burst activity was examined in lavage-procured bovine pulmonary alveolar macrophages. Nonstimulated alveolar macrophages released a minimal quantity of superoxide anion and had small amounts of glucose flux through the pathways of energy metabolism. Nonstimulated cells metabolized substantial amounts of glucose through the hexose monophosphate shunt. Stimulation with opsonized zymosan particles induced a tenfold increase in the release of superoxide anion and a twofold increase in the flux of glucose through the hexose monophosphate shunt and the pathways of energy metabolism. Preliminary observations also indicated that the magnitude of the burst varied between sets of bronchoalveolar cells obtained from the same calf over time.

Effect of estradiol and progesterone on antistaphylococcal activity of neutrophils from ovariectomized mares.

Neutrophils isolated from jugular blood of ovariectomized mares were studied for the effect of estradiol and progesterone on bactericidal activity against Staphylococcus aureus. In experiment 1, neutrophils obtained from 4 mares were tested for bactericidal activity by adding estradiol (43 pg/ml) or progesterone (6.4 ng/ml) to the bactericidal assay. In experiment 2, 3 of the 4 ovariectomized mares were given 2 mg of estradiol, IM, daily for 3 days. Eighteen days after the initial estradiol injection, mares were given 300 mg of progesterone, IM, for 6 days. Neutrophils from these mares were tested for bactericidal activity 4 days after the initial estradiol injection, 17 days after the initial estradiol injection (control), and 7 days after the first progesterone injection. Bactericidal activity was measured at 30 and 120 minutes by counting the number of colony-forming units remaining. Neutrophil antistaphylococcal activity was not altered by adding estradiol and progesterone to the assay or by supplementing ovariectomized mares with estradiol and progesterone (P greater than 0.05).

Isolation and partial characterization of equine alveolar macrophages.

A device was constructed from an equine nasogastric tube, polyethylene tubing, and a 3-way stopcock and used to lavage the lungs of anesthetized ponies. The technique was safe and atraumatic in that 6.4 to 19.7 X 10(7) purified alveolar macrophages were removed from the lungs without harm to the ponies or contamination of the samples with blood. Studies of these highly purified cell suspensions revealed a mean viability of 85% as assessed by eosin dye exclusion with a mean recovery (+/- SD) of 12.5 +/- 4.8 X 10(7) pulmonary alveolar macrophages/pony.

Production of superoxide anion by bovine pulmonary macrophages challenged with soluble and particulate stimuli.

The effects of opsonized zymosan, phorbal myristate acetate, and live Pasteurella haemolytica on superoxide anion production by bovine pulmonary macrophages were determined. The anion responses were dose-dependent for all stimuli, except for unopsonized P haemolytica. The effect of viable P haemolytica on macrophage viability was related to bacterial dosage and the presence of opsonizing antibody. Superoxide responses varied directly with the dose of opsonized live P haemolytica, but indirectly with macrophage viability.

Exogenous estradiol reduces inhibition of luteinizing hormone by estradiol in prepubertal heifers.

The hypothesis that high levels of exogenous estradiol administered to heifers during the prepubertal period would decrease subsequent negative feedback of estradiol on luteinizing hormone (LH) secretion was tested. Fourteen prepubertal heifers were ovariectomized on Day 0. Ovariectomized heifers received either no further treatment (OVX, n = 4), a single estradiol implant on Day 0 (OVXE, n = 5), or the single implant on Day 0 and two additional implants between Days 16 and 30 (OVXE+ E, n = 5). Ten ovary-intact heifers received either no treatment (INT, n = 5) or were administered the two estradiol implants between Days 16 and 30 (INT+ 5, n = 5). Comparison of LH secretion in OVXE to OVXE+E, and in INT to INT+E resulted in significant time-by-treatment interactions (p less than 0.05 for both). As pubertal age approached, mean concentration of LH (p less than 0.05) and pulse frequency (p less than 0.05) increased more rapidly in OVXE+E and INT+E than in OVXE and INT, respectively. Amplitude of LH pulses was unaffected by treatment. When data were standardized to day of puberty in INT and INT+E heifers, mean LH concentration and LH pulse frequency increased as puberty approached in both groups. These data confirm earlier reports indicating that secretion of LH increases gradually as puberty approaches in heifers. It was concluded that administration of estradiol during the prepubertal period hastened the decline in the subsequent negative feedback of estradiol. Precocious puberty was not induced in ovary-intact females.