Cross-sectional study to identify the prevalence of and factors associated with laminitis in UK donkeys.

BACKGROUND: Laminitis causes lameness in donkeys, but its prevalence and factors associated with disease remain uncertain. OBJECTIVES: To determine the prevalence of and identify factors associated with laminitis in donkeys. STUDY DESIGN: Retrospective cross-sectional study. METHODS: All donkeys at the Donkey Sanctuary, UK, October 2015 to March 2019 were included. For animals that had laminitis during this period, age, sex, weight, body condition score, and the onset date and type of each episode (first or recurrent, acute or chronic) were recorded. Additionally, management data, foot lesion score, endocrine data, other medical conditions, occurrence of foot trimming, surgical procedures, diagnostic imaging, behavioural modification therapy or movement between farms within the month prior were noted. Controls were animals that did not experience laminitis during this period and similar data were recorded. Multivariable logistic regression modelling assessed the differences between the control group and laminitis outcome groups (first, all laminitis, acute and chronic episodes). RESULTS: Altogether, 707 animals were included; 364 were control animals; 343 had a first episode of laminitis during the study period, of which 200/343 had no further episodes and 143/343 had recurrent episodes resulting in a total of 512 laminitis episodes and the period prevalence was 48.5% over 42 months. Overall, 180/512 (35%) laminitis episodes were acute and 332/512 (65%) were chronic. Compared with control animals, the laminitic outcome groups were significantly (P < .05) more likely to be younger (first episode), less likely to get extra feed (all four groups) or have an additional medical problem (first episode), and less likely to have undergone dental work, movement, imaging (all four groups) or surgery (first; all laminitis, chronic episodes) in the month preceding the episode. MAIN LIMITATIONS: These results may not be applicable to the wider donkey population. CONCLUSIONS: Laminitis commonly affects donkeys, but factors associated with donkey laminitis differ from those reported in horses.

Emergency Management for Donkeys and Mules.

This article provides an overview of initial assessment and management of common emergency presentations in donkeys and mules. The principles are similar to those in horses (and ponies), but clinicians must be aware of differences in recognition of signs of pain/disease, approach to handling, pharmacology of some drugs, and subtle differences in the physiology and local anatomy in donkeys and mules. The epidemiology of common disease presentations will vary between pet/companion or working/farmed donkeys and mules. Regular dental checks, deworming, vaccination, and monitoring of behavior and quality of life are important aspects of preventive care.

Objective assessment of chronic pain in donkeys using the donkey chronic pain scale (DCPS): A scale-construction study.

Assessment of chronic pain is very important for monitoring and improving welfare and quality of life in donkeys. Freedom from disease and pain is one of the 'five freedoms' underlying animal welfare. The aim of the current study was to develop a pain scale for assessment of chronic pain in donkeys (Donkey Chronic Pain Scale; DCPS), including behavioural and facial expression-related parameters. The scale was applied to 77 donkeys (38 donkeys diagnosed with chronic health problems by means of clinical examination and additional diagnostic procedures and 39 healthy control animals). Animals were assessed twice daily for three consecutive days by two observers that were not masked to the condition of the animals but were unaware of the analgesic treatment regimens. Both composite, facial expression-based and combined DCPS pain scales showed excellent inter-observer reliability (Cronbach's alpha = 0.98, 0.96 and 0.98 respectively; P < 0.001). Individual composite and facial expression-based pain scores and the resulting combined DCPS showed significant differences between donkeys with chronic conditions and control donkeys at all time points (P < 0.001). A DCPS cut-off of 6 showed good sensitivity and specificity (92% and 82.5% respectively) for presence of a chronic painful condition. Facial expression-related parameters separately showed low sensitivity. In conclusion, it is possible to use a composite pain scale for assessment of chronic pain in donkeys, based on behavioural and facial expression-based parameters. Further studies are needed to validate this pain scale before it can be used in veterinary practice.

The Welfare Aggregation and Guidance (WAG) Tool: A New Method to Summarize Global Welfare Assessment Data for Equids.

Animal welfare can be represented by an array of indicators. There is, however, increasing demand for concise welfare assessments that can be easily communicated and compared. Previous methods to aggregate welfare assessments have focused on livestock systems and produced a single welfare score, which may not represent all aspects of welfare. We propose an aggregation method for the recently developed Equid Assessment Research and Scoping (EARS) welfare assessment tool that results in grades for five welfare categories: housing conditions, working conditions, health, nutrition, and behavior. We overcome the problems associated with existing approaches by using a single aggregation method (decision trees) that incorporates the most important welfare indicators in a single step. The process aims to identify equids with the poorest welfare and aid decision-making when allocating resources. We demonstrate its application using a case study of over 6000 equids across Europe and Asia, where equids in India and Pakistan had the poorest welfare status in terms of health (respiratory disease and open wounds) and behavior (signs of fear and distress, and limb tethering practices). We recommend identification of the specific causes of these issues, using either existing detailed welfare data or through issue-specific assessments by an appropriate professional, to guide the development of appropriate interventions and, ultimately, improve equid welfare.

Monitoring Acute Pain in Donkeys with the Equine Utrecht University Scale for Donkeys Composite Pain Assessment (EQUUS-DONKEY-COMPASS) and the Equine Utrecht University Scale for Donkey Facial Assessment of Pain (EQUUS-DONKEY-FAP).

Objective pain assessment in donkeys is of vital importance for improving welfare in a species that is considered stoic. This study presents the construction and testing of two pain scales, the Equine Utrecht University Scale for Donkey Composite Pain Assessment (EQUUS-DONKEY-COMPASS) and the Equine Utrecht University Scale for Donkey Facial Assessment of Pain (EQUUS-DONKEY-FAP), in donkeys with acute pain. A cohort follow-up study using 264 adult donkeys (n = 12 acute colic, n = 25 acute orthopaedic pain, n = 18 acute head-related pain, n = 24 postoperative pain, and n = 185 controls) was performed. Both pain scales showed differences between donkeys with different types of pain and their control animals (p < 0.001). The EQUUS-DONKEY-COMPASS and EQUUS-DONKEY-FAP showed high inter-observer reliability (Cronbach's alpha = 0.97 and 0.94, respectively, both p < 0.001). Sensitivity of the EQUUS-DONKEY-COMPASS was good for colic and orthopaedic pain (83% and 88%, respectively), but poor for head-related and postoperative pain (17% and 21%, respectively). Sensitivity of the EQUUS-DONKEY-FAP was good for colic and head-related pain (75% and 78%, respectively), but moderate for orthopaedic and postoperative pain (40% and 50%, respectively). Specificity was good for all types of pain with both scales (91%-99%). Different types of acute pain in donkeys can be validly assessed by either a composite or a facial expression-based pain scale.

Equid Assessment, Research and Scoping (EARS): The Development and Implementation of a New Equid Welfare Assessment and Monitoring Tool.

The assessment of animal welfare poses numerous challenges, yet an emerging approach is the consolidation of existing knowledge into new frameworks which can offer standardised approaches to welfare assessment across a variety of contexts. Multiple tools exist for measuring the welfare of equids, but such tools have typically been developed for specific contexts. There is no 'one size fits all' which means that resulting datasets are generally non-comparable, creating a barrier to knowledge-sharing and collaboration between the many organisations working to improve equid welfare around the globe. To address this, we developed the Equid Assessment, Research and Scoping (EARS) tool, which incorporates pre-existing validated welfare assessment methods alongside new welfare indicators to deliver a larger and more comprehensive series of welfare indicators than currently exists, creating a single resource that can be used to assess equid welfare in any context. We field-trialled three welfare assessment protocols within the EARS tool, and applied these to welfare assessment of equids in a variety of contexts across nineteen countries. The EARS tool proved a useful, versatile and rapid method for collecting welfare assessment data and we collected 7464 welfare assessments in a period of fifteen months. We evaluate the EARS tool and provide ideas for future development.

Clinical Evaluation and Preventative Care in Donkeys.

Clinical evaluation and preventative care in donkeys should follow similar guidelines as for horses. There are species-specific differences due to the desert-adapted physiology of the donkey. Donkeys are mainly used as pack animals, companions and for production of meat or milk - they may be kept well into old age. Diseases often present late or may go unrecognized leading to poor welfare and quality of life. Basic knowledge of nutrition, blood values, pharmacology and common disease recognition will help veterinarians improve the health and welfare of donkeys.

Respiratory Disorders of the Donkey.

Donkeys suffer from the same respiratory diseases as horses; however, owing to their nonathletic nature many conditions can present in a more advanced state before becoming clinically apparent. Anatomically, their respiratory tract is similar to the horse, with certain species-specific differences that are important to be aware of. Often donkeys do not receive the same level of routine care as horses, so many are not vaccinated against respiratory pathogens such as influenza or herpesviruses. Donkeys can act as a reservoir for certain infectious and parasitic respiratory diseases and the interpretation of diagnostic tests needs to be carried out with caution.

Modulation of equine neutrophil adherence and migration by the annexin-1 derived N-terminal peptide, Ac2-26.

Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived.

CXCL8 attenuates chemoattractant-induced equine neutrophil migration.

The chemokine, CXCL8, is a potent chemoattractant but it has also been shown to attenuate the migratory response of human neutrophils to the bacterial peptide, FMLP; this could lead to retention of cells in infected tissue and, potentially, to enhanced clearance of bacteria. This study has examined the effect of CXCL8 on equine neutrophil migration and adherence in response to PAF and LTB(4), chemoattractants that may play a role in non-infectious inflammatory conditions of the horse associated with neutrophil recruitment to the target tissue. The effects of CXCL8 on PAF- and LTB(4)-induced responses were determined using a ChemoTx plate migration assay and by measuring adhesion to protein-coated plastic. The CXCR1/2 antagonist, SB225002, was used to investigate whether the observed effects were receptor mediated and the role of cAMP was examined by measuring intracellular cAMP following exposure to agonists alone and in combination and by establishing the effect of dibutyryl cAMP on neutrophil migration. CXCL8, LTB(4) and PAF each induced migration and adhesion. Exposure of neutrophils to a combination of CXCL8 and PAF reduced the magnitude of the responses to that of unstimulated cells. In contrast, although the effect was less than additive, the response to co-stimulation with CXCL8 and LTB(4) were not nearly as pronounced. CXCL8 acted in a receptor mediated manner, the attenuation of PAF-induced responses being reversed by SB225002 at a concentration that blocks CXCR2. CXCL8, PAF and LTB(4) alone increased intracellular cAMP. In co-incubation studies, combination of CXCL8 with PAF led to an additive increase in cAMP whereas no increase above that obtained in response to LTB(4) alone was seen. Dibutyryl cAMP significantly reduced neutrophil migration in response to either CXCL8 or PAF alone. These results demonstrate that CXCL8, in addition to being a potent chemoattractant and pro-adhesive molecule for equine neutrophils, is able to attenuate responses to PAF and, to a much lesser extent, LTB(4). This effect, which appears to be CXCR2-mediated and cAMP dependent, could lead in vivo to trapping of cells at sites of inflammation resulting potentially in either enhanced clearance of injurious stimuli or increased local tissue damage by activated cells.

Validating 123I-metaiodobenzylguanidine as a platelet marker for non-invasive imaging in rabbits.

INTRODUCTION: Recent in vitro studies in our laboratory have demonstrated that platelets are necessary for leukocyte recruitment and airway remodelling in models of allergic airway inflammation, and also migrate to lung tissues in response to anti-IgE or relevant allergens in allergic asthma. Non-invasive imaging of platelet migration in vivo would provide a further insight into the roles of platelets in inflammatory diseases such as asthma, and metaiodobenzylguanidine (MIBG) was considered as a suitable platelet marker. METHODS: The kinetics of MIBG uptake into rabbit platelets, the effect of MIBG on platelet function and the effect of platelet activation on MIBG uptake and retention were investigated. MIBG-labelled platelets were administered intravenously into rabbits and the time course of radioactivity in the lung and blood was monitored as a function of stimulation. RESULTS: Following a 4h incubation of MIBG in rabbit PRP, a near maximal MIBG uptake (52.4 ± 20.2%) in platelets occurred. This time point was chosen for subsequent in vitro studies. In vitro platelet function studies showed that MIBG has no effect on ADP or PAF-induced platelet aggregation, PAF-induced thromboxane production or fMLP-induced platelet chemotaxis. However, serotonin showed a significant effect on MIBG uptake and retention, but only at high concentrations. Stimulation of rabbit platelets with ADP and PAF caused a significant release of stored MIBG in vitro. Following i.v. administration of MIBG labelled platelets, the response to i.v. ADP and PAF stimulation was small but significant. DISCUSSION: The release of MIBG from platelets in vivo, particularly following stimulation, leads to high background levels. Therefore, MIBG may have limited utility as a label for imaging platelets in vivo using PET. However, it may be a useful marker in detecting pathological conditions where platelet migration is involved.

Evaluation of the effect of phosphodiesterase on equine platelet activation and the effect of antigen challenge on platelet phosphodiesterase activity in horses with recurrent airway obstruction.

OBJECTIVE: To determine whether expression of equine platelet activation-dependent surface markers is influenced by phospodiesterase (PDE) isoenzyme activity and whether antigen challenge alters platelet PDE activity in horses with recurrent airway obstruction (RAO). ANIMALS: 16 horses. PROCEDURES: 7 healthy horses were used for in vitro experiments, 6 horses with RAO were used for antigen challenge, and 6 healthy horses were used as control animals. Three of the healthy horses had also been used in the in vitro experiments. Effects of PDE inhibition and activation of adenylyl cyclase on CD41/61 and CD62P expression on platelets and platelet-neutrophil aggregate formation in vitro were investigated via flow cytometry. Platelet PDE activity and sensitivity to inhibition of PDE3 and PDE5 isoenzymes were examined in horses with RAO and control horses before and after antigen challenge. RESULTS: Inhibition of PDE or activation of adenylyl cyclase significantly inhibited stimulus-induced expression of CD41/61 and CD62P (by approx 94% and 40%, respectively) and percentage of CD62P positive cells (by approx 30%). Only the PDE3 inhibitor, trequinsin, caused a significant (53%) reduction in platelet-neutrophil aggregate formation. Platelet PDE activity decreased following antigen challenge in RAO-affected horses and control horses. In horses with RAO, a significant increase in sensitivity of platelet PDE to inhibition by the PDE5 inhibitor zaprinast was observed after 5 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Results provided further evidence that PDE3 is an important regulator of equine platelet activation and suggested that changes in regulation of platelet PDE5 may contribute to antigen-induced response in horses with RAO.

Winter body mass and over-ocean flocking as components of danger management by Pacific dunlins.

BACKGROUND: We compared records of the body mass and roosting behavior of Pacific dunlins (Calidris alpina pacifica) wintering on the Fraser River estuary in southwest British Columbia between the 1970s and the 1990s. 'Over-ocean flocking' is a relatively safe but energetically-expensive alternative to roosting during the high tide period. Fat stores offer protection against starvation, but are a liability in escape performance, and increase flight costs. Peregrine falcons (Falco peregrinus) were scarce on the Fraser River estuary in the 1970s, but their numbers have since recovered, and they prey heavily on dunlins. The increase has altered the balance between predation and starvation risks for dunlins, and thus how dunlins regulate roosting behavior and body mass to manage the danger. We therefore predicted an increase in the frequency of over-ocean flocking as well as a decrease in the amount of fat carried by dunlins over these decades. RESULTS: Historical observations indicate that over-ocean flocking of dunlins was rare prior to the mid-1990s and became common thereafter. Residual body masses of dunlins were higher in the 1970s, with the greatest difference between the decades coinciding with peak peregrine abundance in October, and shrinking over the course of winter as falcon seasonal abundance declines. Whole-body fat content of dunlins was lower in the 1990s, and accounted for most of the change in body mass. CONCLUSIONS: Pacific dunlins appear to manage danger in a complex manner that involves adjustments both in fat reserves and roosting behavior. We discuss reasons why over-ocean flocking has apparently become more common on the Fraser estuary than at other dunlin wintering sites.

Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production.

N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil superoxide production was measured by the reduction of cytochrome (cyt) C following stimulation with Ac2-26 and the formyl peptide receptor (FPR) agonists, FMLP, WKYMVm and WKYMVM. Responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in agonist-induced effects was determined using PD98059. The effect of Ac2-26 on superoxide production in response to serum-treated zymosan (STZ) was also investigated, and the roles of FPR and p42/44 MAPK ascertained. Annexin-1 was detected in both equine neutrophils and mononuclear cells using a polyclonal rabbit anti-human annexin-1 antibody. Ac2-26 (5x10(-5)M) induced superoxide production in cytochalasin B-primed (48+/-8 versus 21+/-9 (unstimulated cells) nmol cyt C/10(6) neutrophils) and un-primed cells (37+/-10 versus 11+/-5 nmol cyt C/10(6) neutrophils). FMLP and WKYMVm, but not WKYMVM, also caused superoxide production in primed neutrophils, suggesting the response was mediated by FPR receptor binding. This was supported by the marked inhibitory effect of BOC-2 on the responses to Ac2-26 and FMLP although, interestingly, the effects of WKYMVm were not significantly reduced (50+/-5 (WKYMVm) versus 45+/-5 (WKYMVm+BOC-2) nmol reduced cyt C/10(6) neutrophils). Inhibition of p42/44 MAPK activation with PD98059 significantly attenuated superoxide production in response to Ac2-26, FMLP and WKYMVm and Western blotting showed that Ac2-26 induced p42/44 MAPK activation. At a concentration which did not cause superoxide production, Ac2-26 (10(-5)M) significantly reduced the response to STZ (84+/-17% inhibition). This inhibitory effect was attenuated by both BOC-2 and PD98059. These results suggest that if activation of equine leucocytes in vivo leads to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and decrease free radical production in response to particulate stimuli. This could help to reduce local tissue damage but, as Ac2-26 can also stimulate superoxide production at higher concentrations in an FPR-dependent manner, the amount of free radical production may depend on the concentration of peptide present.

Phosphodiesterase isoenzymes in equine platelets and their influence on platelet adhesion.

OBJECTIVE: To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion. SAMPLE POPULATION: Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean +/- SEM, 17.3 +/- 1.1 years). PROCEDURES: PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay. RESULTS: PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PDE3 abolished platelet adhesion almost completely, whereas inhibition of PDE4 and PDE5 had little effect. CONCLUSIONS AND CLINICAL RELEVANCE: Function of equine platelets can be influenced by inhibition of PDE3. Selective PDE3 inhibitors may be clinically useful to regulate platelet function. They offer the advantage of increased potency with fewer adverse effects, compared with those for nonselective PDE inhibitors.