Changes of adenosine deaminase activity in serum and saliva around parturition in sows with and without postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) is associated with a significantly higher activation of the inflammatory and stress response at parturition than in the healthy sow. Therefore, reliable and possibly non-invasive biomarkers for substantial increases of inflammation are searched to support the PDS diagnosis. This report studies the possible changes of the inflammatory marker enzyme adenosine deaminase (ADA) in serum and saliva of 38 PDS positive sows (PDS+) and 38 healthy sows (PDS-). Sampling was performed every 24 h from 60 h before to 36 h after parturition. Isoenzyme 1 (ADA1) and isoenzyme 2 (ADA2), as well as total ADA (tADA), were measured and their statistical association with several serum and saliva biomarkers of inflammation and stress was investigated. RESULTS: Compared to a baseline (60 to 36h prepartum), salivary activities of ADA1, ADA2 and tADA increased significantly over time in both PDS+ and PDS- sows, reaching their peaks after parturition. In serum from PDS- sows, no changes were observed over time in either ADA1, ADA2 or tADA. In PDS+ sows, serum ADA2 activity decreased temporarily after parturition followed by a significant increase compared to baseline. ADA1, ADA2 and tADA were all significantly associated with several inflammatory biomarkers and ADA1 in serum was associated with serum cortisol. Although serum activity was higher in PDS+ than in PDS- sows, the differences were not statistically significant. Further, no difference was noted between the groups in the analyses of saliva. CONCLUSIONS: Salivary ADA1 and ADA2 increased in all sows after parturition, potentially as a response to the postpartum inflammation. However, no difference in the activity of ADA1, ADA2 and tADA were found between PDS+ and PDS- sows indicating inability to diagnose PDS under the conditions described in this report.

Inflammatory markers before and after farrowing in healthy sows and in sows affected with postpartum dysgalactia syndrome.

BACKGROUND: The pathogenesis of postpartum dysgalactia syndrome (PDS) in sows is not fully elucidated and affected sows often present vague clinical signs. Accurate and timely diagnosis is difficult, and PDS is often recognized with a delay once piglets begin to starve. Increased rectal temperature of the sow is an important diagnostic parameter, but it may also be influenced by a number of other parameters and is thus difficult to interpret. Inflammatory markers may be important adjuncts to the clinical assessment of sows with PDS, but such markers have only been studied to a limited extent. The objective was to characterize the inflammatory response in healthy sows and in sows suffering from PDS, and to identify biomarkers that may assist in early identification of PDS-affected sows. RESULTS: Thirty-eight PDS-affected (PDS+) and 38 healthy (PDS-) sows underwent clinical examination and blood sampling every 24 h, from 60 h before the first piglet was born to 36 h after parturition. In both groups, inflammatory markers changed in relation to parturition. Most inflammatory markers changed 12-36 h after parturition [white blood cell counts (WBC), neutrophil counts, lymphocyte counts, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), serum amyloid A (SAA), C-reactive protein (CRP), haptoglobin (Hp), iron (Fe) and albumin (ALB)]. Changes in neutrophil counts, lymphocyte counts, CRP, Fe and ALB were observed -12 to 0 h before parturition. WBC, neutrophil and lymphocyte counts, serum concentrations of TNF-α, IL-6, Hp and Fe differed between PDS+ and PDS- sows. These differences were mainly apparent 12 to 36 h after parturition, but already at 12 h before parturition, PDS+ sows had lower lymphocyte counts than PDS- sows. CONCLUSIONS: Parturition itself caused significant inflammatory changes, but PDS+ sows showed a more severe response than PDS- sows. WBC, neutrophil and lymphocyte counts, and concentrations of TNF-α, IL-6, Hp and Fe can be potential biomarkers for PDS. Lymphocyte counts may be used to detect PDS at pre-partum. To assess their diagnostic potential, these markers must be investigated further and most likely combined with assessment of clinical parameters and other biomarkers for improved identification of sows at risk of developing PDS.

Hormonal and metabolic indicators before and after farrowing in sows affected with postpartum dysgalactia syndrome.

BACKGROUND: Postpartum dysgalactia syndrome (PDS) in sows is difficult to diagnose and the pathogenesis is obscure. Hormonal changes related to the disease are often difficult to distinguish from those found in the normal transition period from gestation to lactation. The study aimed to investigate metabolic and hormonal changes related to PDS with the goal of identifying potential biomarkers in sows suffering from PDS (PDS+). Selected biomarkers were examined by comparing 38 PDS+ sows with 38 PDS negative (PDS-) sows. The sows were sampled every 24 h from 60 h ante partum (a.p.) to 36 h post partum (p.p.). RESULTS: Compared to the baseline (60 to 36 h a.p.), cortisol in serum and saliva and fasting blood glucose concentrations increased in PDS+ as well as PDS- sows. C-peptide decreased relative to the baseline in PDS+ sows, and prolactin and 8-epi prostaglandin F2 alpha (8-epi-PGF2α) decreased in PDS- sows. Concentrations of cortisol in serum and saliva, salivary chromogranin A (CgA), fasting blood glucose, C-peptide, and 8-epi-PGF2α differed significantly between PDS+ and PDS- sows, with levels of cortisol in serum and saliva, salivary CgA, and 8-epi-PGF2α in serum being different in the two groups already before parturition. Concentrations of salivary CgA were significantly lower in PDS- sows than in PDS+ sows during the entire study period. CONCLUSIONS: The results suggest that salivary CgA, cortisol and serum 8-epi-PGF2α may potentially serve as early diagnostic indicators for PDS. The consistently higher salivary CgA concentration in PDS+ sows compared to PDS- sows may indicate that homeostatic disturbances are present between 36 to 60 h before parturition in sows developing PDS. The higher serum and saliva cortisol concentration in PDS+ sows compared to PDS- sows could reflect an early sign of inflammation or stress. The significantly lower C-peptide in PDS+ sows compared to PDS- sows may reflect a lower food intake. Our results contribute to the understanding of the pathogenesis of PDS, and the homeostatic disturbances detected before parturition warrants further investigation. The diagnostic potential of the markers identified in this study should be investigated further in a larger population of sows.

Changes in concentrations of haemostatic and inflammatory biomarkers in synovial fluid after intra-articular injection of lipopolysaccharide in horses.

BACKGROUND: Septic arthritis is a common and potentially devastating disease characterized by severe intra-articular (IA) inflammation and fibrin deposition. Research into equine joint pathologies has focused on inflammation, but recent research in humans suggests that both haemostatic and inflammatory pathways are activated in the joint compartment in arthritic conditions. The aim of this study was to characterize the IA haemostatic and inflammatory responses in horses with experimental lipopolysaccharide (LPS)-induced joint inflammation. Inflammation was induced by IA injection of LPS into one antebrachiocarpal joint of six horses. Horses were evaluated clinically with subjective grading of lameness, and blood and synovial fluid (SF) samples were collected at post injection hours (PIH) -120, -96, -24, 0, 2, 4, 8, 16, 24, 36, 48, 72 and 144. Total protein (TP), white blood cell counts (WBC), serum amyloid A (SAA), haptoglobin, iron, fibrinogen, thrombin-antithrombin (TAT) and d-dimer concentrations were assessed in blood and SF. RESULTS: Intra-articular injection of LPS caused local and systemic signs of inflammation including increased rectal temperature, lameness and increased joint circumference and skin temperature. Most of the biomarkers (TP, WBC, haptoglobin, fibrinogen and TAT) measured in SF increased quickly after LPS injection (at PIH 2-4), whereas SAA and d-dimer levels increased more slowly (at PIH 16 and 144, respectively). SF iron concentrations did not change statistically significantly. Blood WBC, SAA, haptoglobin and fibrinogen increased and iron decreased significantly in response to the IA LPS injection, while TAT and d-dimer concentrations did not change. Repeated pre-injection arthrocenteses caused significant changes in SF concentrations of TP, WBC and haptoglobin. CONCLUSION: Similar to inflammatory joint disease in humans, joint inflammation in horses was accompanied by an IA haemostatic response with changes in fibrinogen, TAT and d-dimer concentrations. Inflammatory and haemostatic responses were induced simultaneously and may likely interact. Further studies of interactions between the two responses are needed for a better understanding of pathogenesis of joint disease in horses. Knowledge of effects of repeated arthrocenteses on levels of SF biomarkers may be of value when markers are used for diagnostic purposes.

Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach.

Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome, we compared the contents of different fractions from bovine colostrum and milk from our 2 previous studies. A total of 140 colostrum fluid-phase proteins and 103 milk fluid-phase proteins were detected. In the cellular fraction, 324 and 310 proteins were detected in colostrum and milk, respectively. In total, 514 proteins were detected, of which 162 were in the fluid phase. Of these, 50 proteins were exclusively seen in colostrum, 13 were exclusively seen in milk, and 99 were common to colostrum and milk. Ranking of proteins mutually and calculating protein ratios based on spectral counts and ELISA resulted in new information on how proteins were associated with the fluid or cellular fraction of the samples. Interestingly, despite lower counts/concentrations than the classical proteins such as immunoglobulins, ß-lactoglobulin, and lactotransferrin, several proteins appeared in higher or similar colostrum:milk spectral count ratios as these. Using this approach indicated, for example, that osteopontin, haptoglobin, milk amyloid A, and gelsolin may be interesting molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.

Equine deep stromal abscesses (51 cases - 2004-2009)--Part 2: the histopathology and immunohistochemical aspect with attention to the histopathologic diagnosis, vascular response, and infectious agents.

PURPOSE: To investigate histopathologic and immunohistochemical aspects of equine deep stromal abscesses (DSA) with a focus on the histopathologic diagnosis, presumptive etiology, and the immunohistochemical expression of three angiogenesis-related factors: vascular endothelial growth factor-A (VEGF-A), pigment epithelium-derived factor (PEDF), and interleukin-1 receptor antagonist (IL-1ra). SAMPLE POPULATION: Paraffin-embedded biopsy samples from 51 DSA. The biopsies were collected from full-thickness penetrating keratoplasty or split-thickness lamellar keratoplasty surgeries at the University of Florida Veterinary Medical Center in the period from 2004 to 2009. PROCEDURE: The histopathologic and immunohistochemical findings were tested for association between each other. Prevalence calculation and test for association with qualitative data analysis was used for data evaluation. RESULTS: Fungal hyphae were found histologically in 47.1% (n = 24) of the DSA cases. Histopathologically, most fungal DSA showed suppurative keratitis (n = 34; 66.7%) and little to no stromal vascularization infiltrating the abscess (negative association, P = 0.005). All three angiogenesis-related factors were expressed to some degree in DSA tissue. A negative association between VEGF-A and PEDF when compared to the presence of fungal hyphae (P < 0.001, P = 0.023) indicated that cases positive for these two factors will most probably not have fungal hyphae present. CONCLUSION: Abnormally decreased VEGF-A expression is suggested as the reason for the slow vascularization and delayed resolution of fungal DSA, whereas PEDF and IL-ra did not seem to have any influence on the vascularization process. Clinical and histopathologic characteristics of DSA make it possible to suggest an etiology for an equine DSA with an unknown etiology.

Changes in the equine facial repertoire during different orthopedic pain intensities.

A number of facial expressions are associated with pain in horses, however, the entire display of facial activities during orthopedic pain have yet to be described. The aim of the present study was to exhaustively map changes in facial activities in eight resting horses during a progression from sound to mild and moderate degree of orthopedic pain, induced by lipopolysaccharides (LPS) administered in the tarsocrural joint. Lameness progression and regression was measured by objective gait analysis during movement, and facial activities were described by EquiFACS in video sequences (n = 348, total length 892.5 min) of the horses obtained when resting in their box stalls. Predictive modeling identified 16 action units and action descriptors, related to ears, eyes, and lower face. Lower lip depressor (AU16), lips part (AU25), half blink (AU47), single ear forward (SEAD101) and single ear rotator (SEAD104) were selected as co-occurring significantly more in horses with pain than in horses without pain. The major change in co-occurring facial activities occurred in the transition from no pain to mild pain. In conclusion, resting horses with induced orthopedic pain showed a dynamic upper and lower facial repertoire and the relationship between level of pain intensity and facial activity appears complex.

Is Markerless More or Less? Comparing a Smartphone Computer Vision Method for Equine Lameness Assessment to Multi-Camera Motion Capture.

Computer vision is a subcategory of artificial intelligence focused on extraction of information from images and video. It provides a compelling new means for objective orthopaedic gait assessment in horses using accessible hardware, such as a smartphone, for markerless motion analysis. This study aimed to explore the lameness assessment capacity of a smartphone single camera (SC) markerless computer vision application by comparing measurements of the vertical motion of the head and pelvis to an optical motion capture multi-camera (MC) system using skin attached reflective markers. Twenty-five horses were recorded with a smartphone (60 Hz) and a 13 camera MC-system (200 Hz) while trotting two times back and forth on a 30 m runway. The smartphone video was processed using artificial neural networks detecting the horse's direction, action and motion of body segments. After filtering, the vertical displacement curves from the head and pelvis were synchronised between systems using cross-correlation. This rendered 655 and 404 matching stride segmented curves for the head and pelvis respectively. From the stride segmented vertical displacement signals, differences between the two minima (MinDiff) and the two maxima (MaxDiff) respectively per stride were compared between the systems. Trial mean difference between systems was 2.2 mm (range 0.0-8.7 mm) for head and 2.2 mm (range 0.0-6.5 mm) for pelvis. Within-trial standard deviations ranged between 3.1-28.1 mm for MC and between 3.6-26.2 mm for SC. The ease of use and good agreement with MC indicate that the SC application is a promising tool for detecting clinically relevant levels of asymmetry in horses, enabling frequent and convenient gait monitoring over time.

Accuracy and precision of equine gait event detection during walking with limb and trunk mounted inertial sensors.

The increased variations of temporal gait events when pathology is present are good candidate features for objective diagnostic tests. We hypothesised that the gait events hoof-on/off and stance can be detected accurately and precisely using features from trunk and distal limb-mounted Inertial Measurement Units (IMUs). Four IMUs were mounted on the distal limb and five IMUs were attached to the skin over the dorsal spinous processes at the withers, fourth lumbar vertebrae and sacrum as well as left and right tuber coxae. IMU data were synchronised to a force plate array and a motion capture system. Accuracy (bias) and precision (SD of bias) was calculated to compare force plate and IMU timings for gait events. Data were collected from seven horses. One hundred and twenty three (123) front limb steps were analysed; hoof-on was detected with a bias (SD) of -7 (23) ms, hoof-off with 0.7 (37) ms and front limb stance with -0.02 (37) ms. A total of 119 hind limb steps were analysed; hoof-on was found with a bias (SD) of -4 (25) ms, hoof-off with 6 (21) ms and hind limb stance with 0.2 (28) ms. IMUs mounted on the distal limbs and sacrum can detect gait events accurately and precisely.

Performance of four equine pain scales and their association to movement asymmetry in horses with induced orthopedic pain.

Objective: This study investigated the relationship between orthopedic pain experienced at rest, and degree of movement asymmetry during trot in horses with induced reversible acute arthritis. Orthopedic pain was assessed with the Horse Grimace Scale (HGS), the Equine Utrecht University Scale of Facial Assessment of Pain (EQUUS-FAP), the Equine Pain Scale (EPS), and the Composite Orthopedic Pain Scale (CPS). Reliability and diagnostic accuracy were evaluated with intraclass correlation coefficients (ICC) and area under the curve (AUC). Study design and animals: Eight healthy horses were included in this experimental study, with each horse acting as its own control. Methods: Orthopedic pain was induced by intra-articular lipopolysaccharide (LPS) administration. Serial pain assessments were performed before induction and during pain progression and regression, where three observers independently and simultaneously assessed pain at rest with the four scales. Movement asymmetry was measured once before induction and a minimum of four times after induction, using objective gait analysis. Results: On average 6.6 (standard deviation 1.2) objective gait analyses and 12.1 (2.4) pain assessments were performed per horse. The ICC for each scale was 0.75 (CPS), 0.65 (EPS), 0.52 (HGS), and 0.43 (EQUUS-FAP). Total pain scores of all scales were significantly associated with an increase in movement asymmetry (R 2 values ranging from -0.0649 to 0.493); with CPS pain scores being most closely associated with movement asymmetry. AUC varied between scales and observers, and CPS was the only scale where all observers had a good diagnostic accuracy (AUC > 0.72). Conclusions and clinical relevance: This study identified significant associations between pain experienced at rest and degree of movement asymmetry for all scales. Pain scores obtained using CPS were most closely associated with movement asymmetry. CPS was also the most accurate and reliable pain scale. All scales had varying linear and non-linear relations between total pain scores and movement asymmetry, illustrating challenges with orthopedic pain assessment during rest in subtly lame horses since movement asymmetry needs to be rather high before total pain score increase.

Dynamics of local gene regulations in synovial fluid leukocytes from horses with lipopolysaccharide-induced arthritis.

The role of resident cells such a synoviocytes and chondrocytes in intra-articular inflammation is well-characterized, however the in vivo gene expression patterns of cells (predominantly leukocytes) in the synovial fluid (SF) of an inflamed joint have never previously been investigated. The aim of this study was to investigate gene expression in SF leukocytes from the inflamed joint cavity after intra-articular lipopolysaccharide (LPS) injection in horses to improve our understanding of the temporal regulation of the intra-articular inflammatory response. Gene expression was investigated in SF samples available from six horses 2, 4, 8 16 and 24 h after experimental induction of inflammation in the radiocarpal joint by lipopolysaccharide (LPS) injection. Leukocytic expression of 43 inflammation-related genes was studied using microfluidic high throughput qPCR (Fluidigm®). Expression of 26 genes changed significantly over the 24 h study period, including pro- and anti-inflammatory genes such as interleukin (IL)1, IL6, tumor necrosis factor (TNF), cyclooxygenase 2 (COX2), IL1 receptor antagonist (IL1RN), IL10, and superoxide dismutase 2 (SOD2), chemokine genes, apoptosis-related genes, and genes related to cartilage turnover (matrix metalloproteinase 8 and tissue inhibitor of metalloproteinase 1). The inflammatory responses appeared to be regulated, as an early increase (at 2 h) in expression of the pro-inflammatory genes IL1, IL6, TNF and COX2 was rapidly followed by increased expression (at 4 h) of several anti-inflammatory genes (IL10, IL1RN and SOD2). Similarly, both pro- and anti-apoptotic gene expression as well as expression of chondrodegenerative and chondroprotective genes were activated in SF leukocytes. Thus, the inflammatory response in leukocytes infiltrating the joint in the acute stage of arthritis was well orchestrated in this single-hit LPS-induced arthritis model. This study is the first to describe gene expression patterns in SF-derived leukocytes in vivo during severe joint inflammation, and the results thus expand our knowledge of basic inflammatory mechanisms in the early local response in an inflamed joint.

Towards Machine Recognition of Facial Expressions of Pain in Horses.

Automated recognition of human facial expressions of pain and emotions is to a certain degree a solved problem, using approaches based on computer vision and machine learning. However, the application of such methods to horses has proven difficult. Major barriers are the lack of sufficiently large, annotated databases for horses and difficulties in obtaining correct classifications of pain because horses are non-verbal. This review describes our work to overcome these barriers, using two different approaches. One involves the use of a manual, but relatively objective, classification system for facial activity (Facial Action Coding System), where data are analyzed for pain expressions after coding using machine learning principles. We have devised tools that can aid manual labeling by identifying the faces and facial keypoints of horses. This approach provides promising results in the automated recognition of facial action units from images. The second approach, recurrent neural network end-to-end learning, requires less extraction of features and representations from the video but instead depends on large volumes of video data with ground truth. Our preliminary results suggest clearly that dynamics are important for pain recognition and show that combinations of recurrent neural networks can classify experimental pain in a small number of horses better than human raters.

The pharmacokinetics of the weakly protein-bound anionic compound diatrizoate in serum and synovial fluid of the horse.

PURPOSE: To establish a pharmacokinetic model for the model drug, sodium diatrizoate (DTZ), allowing joint disappearance kinetics to be estimated from serum appearance kinetics following intra-articular administration, and to calculate the relative joint exposure after intravenous and intra-articular DTZ administration (F(iv/IA)). METHODS: Each of five horses received an aqueous solution of 3.9 mg/kg sodium diatrizoate both intravenously and intra-articularly separated by a one-week wash out period. Serum and synovial samples were collected over 7 h and analyzed for content of model compound using inductively coupled plasma mass spectrometry. RESULTS: Differential equations were used for describing the transport of DTZ between the joint and the central compartment. The three-compartment lag-time model obtained demonstrates that the rate of drug appearance in the systemic circulation equals the rate of disappearance from the joint compartment. Following intravenous and intra-articular administration, an average F(iv/IA) of 0.04% (n = 4) was calculated based on the synovial fluid profiles of DTZ. CONCLUSIONS: This study implies that aspects of the intra-articular fate of DTZ can be obtained from serum data in case synovial fluid samplings are limited, for various possible reasons. The low F(iv/IA) may stimulate future research in the field of intra-articular administration of anti-osteoarthritic drugs.

Pharmacokinetics of intra-articular morphine in horses with lipopolysaccharide-induced synovitis.

OBJECTIVE: To describe the pharmacokinetics of intra-articularly (IA) administered morphine. STUDY DESIGN: Experimental randomized, cross-over study. ANIMALS: Eight adult healthy mixed breed horses aged 6.5 +/- 2.3 (mean +/- SD) years and weighing 535 +/- 86 kg. METHODS: Unilateral radiocarpal synovitis was induced by IA injection of 3 microg lipopolysaccharide (LPS) on two occasions (right and left radiocarpal joint, respectively) separated by a 3-week wash-out period. Treatments were administered 4 hours post-LPS-injection: Treatment IA; preservative free morphine IA (0.05 mg kg(-1)) plus saline intravenous (IV) and treatment IV; saline IA plus preservative free morphine IV (0.05 mg kg(-1)). Concentrations of morphine, morphine-3-glucuronide and morphine-6-glucuronide (M6G) were determined repeatedly in serum and synovial fluid (SF) by high-performance liquid chromatography mass spectrometry, at 2 and 4 hours and then at 4 hours intervals until 28 hours post-treatment. RESULTS: Injection of LPS elicited a marked and comparable synovitis in all LPS-injected radiocarpal joints. IA administered morphine was detectable in SF of all eight joints 24 hours post-treatment and in 6/8 joints 28 hours post-treatment. The terminal half-life of morphine in SF was estimated to be 2.6 hours. IA administration of morphine resulted in mean serum concentrations of morphine below 5 ng mL(-1) from 2 to 28 hours after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Intra-articularly administered morphine remained within the joint for at least 24 hours. At the same time only very low serum concentrations of morphine and M6G were detected. The present results suggest that IA morphine at 0.05 mg kg(-1) may be used for IA analgesia lasting at least 24 hours and give strong support to the theory that previously observed analgesic and anti-inflammatory effects of IA morphine in horses are most likely to be mediated peripherally.

Equine Facial Action Coding System for determination of pain-related facial responses in videos of horses.

During the last decade, a number of pain assessment tools based on facial expressions have been developed for horses. While all tools focus on moveable facial muscles related to the ears, eyes, nostrils, lips, and chin, results are difficult to compare due to differences in the research conditions, descriptions and methodologies. We used a Facial Action Coding System (FACS) modified for horses (EquiFACS) to code and analyse video recordings of acute short-term experimental pain (n = 6) and clinical cases expected to be in pain or without pain (n = 21). Statistical methods for analyses were a frequency based method adapted from human FACS approaches, and a novel method based on co-occurrence of facial actions in time slots of varying lengths. We describe for the first time changes in facial expressions using EquiFACS in video of horses with pain. The ear rotator (EAD104), nostril dilation (AD38) and lower face behaviours, particularly chin raiser (AU17), were found to be important pain indicators. The inner brow raiser (AU101) and eye white increase (AD1) had less consistent results across experimental and clinical data. Frequency statistics identified AUs, EADs and ADs that corresponded well to anatomical regions and facial expressions identified by previous horse pain research. The co-occurrence based method additionally identified lower face behaviors that were pain specific, but not frequent, and showed better generalization between experimental and clinical data. In particular, chewing (AD81) was found to be indicative of pain. Lastly, we identified increased frequency of half blink (AU47) as a new indicator of pain in the horses of this study.

Effect of work on body language of ranch horses in Brazil.

The horses' responses to exercise are commonly monitored using physiological variables, nonetheless physical and mental states can also be expressed through body language. The aims of this study were: (i) to identify how facial expressions and other behavioural variables change in ranch horses after a routine workday, and (ii) to investigate if these changes can be used as indicators of physical tiredness by relating them to known variables of physical fitness and workload. Fourteen crossbred ranch horses were assessed pre- and post-workday on two farms, recording the body language, physiological and workload variables. Statistical analysis consisted of four stages: (i) comparisons between the sampling times (pre- vs post-workday) using linear mixed-effects models with repeated measures and a paired Wilcoxon test; (ii) selection of the most powerful variables by applying Kaiser-Meyer-Olkin test and principal components analyses (PCA); (iii) evaluations of the relationships within these selected variables utilizing PCA and Spearman rank coefficients; and (iv) identifying a critical level of the most robust behavioural indicators using a non-hierarchical cluster analysis. Results showed that after a workday the horses increased the frequency/duration of body language indicative of resting. They also decreased the frequency/duration of body language indicative of attention and movements to avoid flies. However, some of these behaviours are also shown when horses are in pain, leading us to suggest that some ranch horses were probably experiencing a combination of of tiredness and slight soreness. Of particular interest, because of the ease with which it can be assessed on the farm and generalized to other situations, we suggest that the frequency of shifting weight between the forelegs has potential to be used as an indicator of physical tiredness in horses. The results can also be used in the development of tools to improve the welfare of ranch horses as well as horses used in other activities, although more research is needed to validate this assumption.

Neuromas at the castration site in geldings.

BACKGROUND: Inguinal pain, unexplained hind limb lameness, back pain or behavioural problems in geldings could be attributable to painful neuromas that develop as a consequence of crushing and severing the testicular nerves during castration. The presence of neuroma in this anatomical location has never been reported, hence the knowledge of possible clinical relevance is limited. The aim of this study was to histologically investigate the testicular nerves at the castration site in geldings for the presence of neuromas. Proximal spermatic cord remnants were collected from 20 geldings admitted to routine post mortem examination for various reasons. The time of castration was unknown, but it had not been performed during the last year. Spermatic cord specimens were immersed in 10% formalin, trimmed, dehydrated, embedded in paraffin, sectioned and stained with haematoxylin and eosin (HE) for light microscopy. Identification of nerve tissue was done by immuno-localization of nerve specific enolase (NSE). RESULTS: Neuromas were found in 21 spermatic cords from 13 geldings and were bilateral in eight of the horses. The neuromas consisted of areas with small groups of non-neoplastic proliferations of peripheral neural tissue. The tissue included neurofilaments and Schwann cells, intermingled or surrounded with, epineural, perineural and endoneural fibrous tissue. The neural tissue immunostained positive with NSE. CONCLUSIONS: This study showed neuromas of the remnant testicular nerves at the site of castration. Further studies are required to establish if these neuromas in the castration site are painful and if certain castration methods promote their formation. Future studies should also investigate the clinical consequence of these neuromas for the individual horse.

Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis.

The aim of the study was to determine the intraarticular serum amyloid A (SAA) response pattern in horses with inflammatory arthritis. Inflammatory arthritis was induced by injection of lipopolysaccharide (LPS) into the radiocarpal joint of four horses. Serum and synovial fluid (SF) samples were collected before and at 4, 8, 12, 24, 48, 72, 96, and 144 h after injection. Concentrations of SAA were measured by immunoturbidometry, and expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. The LPS injection caused systemic and local clinical signs of inflammation. Serum amyloid A appeared in serum and SF within 8h after LPS injection. Isoelectric focusing showed three major SAA bands with apparent isoelectric points (pI) of 7.9, 8.6, and >9.3 in serum and SF. Synovial fluid contained two additional isoforms with highly alkaline apparent pI values (apparent pI value extrapolated from standard curve=10.0 and 10.2), which were not present in any of the serum samples. In conclusion, intraarticular injection of LPS induced systemic and local inflammatory responses in the horses. By demonstrating SF-specific SAA isoforms the results of the present study suggest that SAA is synthesized locally in the equine inflamed joint, similar to what has been demonstrated in humans previously. The marked local SAA synthesis suggests an important pathophysiological role in inflammatory arthritis.

The use of liquid chromatography tandem mass spectrometry to detect proteins in saliva from horses with and without systemic inflammation.

The objective of the study was to assess global expression of proteins in equine saliva using liquid chromatography tandem mass spectrometry (LC-MS/MS). Saliva was obtained from seven horses with and six horses without evidence of systemic inflammatory disease. Tryptic peptides from saliva were analysed by LC-MS/MS. Of 195 unique proteins identified, 57 were detected only in saliva samples from horses with systemic inflammation (in two to six of the seven horses). Among the differentially expressed proteins were several acute phase proteins (APPs) such as serum amyloid A, fibrinogen, haptoglobin, and alpha1-acid glycoprotein. The study is the first to describe detection of inflammatory proteins in horse saliva. The proteins detected were similar to those described in saliva from cattle, small ruminants and pigs. Detection of APPs in horses with systemic inflammation suggests that saliva may be used for non-invasive disease monitoring in horses as in humans, pigs and dogs.

Monitoring of equine health in Denmark: a survey of the attitudes and concerns of potential database participants.

At present Denmark has no central database holding records of equine health and disease. Nor have attitudes to the establishment of a national database, and the concerns it raises, been investigated in a systematic way. The objective of the present study was to assess the attitudes and concerns of potential database stakeholders. Attitudes to participation, and the location, financing and management of the proposed database, together with any concerns it raises, were investigated in a questionnaire study of 13 potential stakeholder groups in Denmark (in total, 1581 questionnaire recipients and 717 respondents): equine veterinarians, researchers, veterinary students, animal welfare organization representatives, horse owners, trainers, farriers, representatives from authorities, ordinary citizens, and representatives of laboratories, and insurance, medical equipment and pharmaceutical companies. Proportions were calculated for pre-categorised responses, and supplementary attitudes were extracted from qualitative responses. Eighty-six percent of respondents stated a positive interest in providing data for the database; the percentage for veterinarians was 90%. Data contribution was regarded as feasible by many of the stakeholder groups; this willingness to provide data, however, depended on the implementation of a very user-friendly system. Requirements included an electronic, simple, and time-efficient data reporting system. Most respondents felt the database should be financed via horse owners through mandatory contributions linked to each horse. Disagreement appeared to arise over data ownership, accessibility, and location. These and other issues need further elaboration in order to find a solution that is acceptable for all stakeholders. It may be advisable to collect and store data in more than one database.