DS research

Donkeys and horses share several parasites including the small strongyles, Cyathostominae. Moxidectin (MOX), a compound of macrocyclic lactones, has a wide range of ecto and endoparasitic activity in many species. For horses, MOX is available as oral gel formulation that provides excellent and long-lasting efficacy against nematodes such as large and small strongyles. There is a paucity of data available on the efficacy of anthelmintics used in donkeys (Veneziano et al., 2011). Therapeutics, such as antiparasitic compounds, are often administered to donkeys on the basis of dosage and intervals recommended for horses, because very few drugs have donkey-specific label indications (Grosenbaugh et al., 2011). The objective of the present study was to evaluate the field efficacy and Egg Reappearance Period (ERP) of MOX oral gel up to 84 days at horse dose against natural infection of Cyathostominae in donkeys.

Introduction

Pleuroparenchymal Fibroelastosis (PPFE) is an emerging, idiopathic and likely under diagnosed condition that does not fall within any of the current classifications of human interstitial lung diseases (Frankel et al, 2004). Key features include an upper zone predominance of pleural fibrosis with associated intra-alveolar fibrosis and elastosis of alveolar walls (Frankel et al, 2004; Reddy et al., 2012). The objective of our study was to examine ex vivo lung tissue from a small cohort of aged donkeys with a high prevalence (35%) of fibrosing interstitial lung disease termed Asinine Pulmonary Fibrosis (APF;Morrow et al, 2010). APF is a potentially debilitating and untreatable syndrome of donkeys that is poorly understood and rarely documented. We hypothesise that some cases of APF share several key gross and histopathological features of PPFE and propose that both can be linked to recurrent respiratory infection.

Materials and methods

Whole asinine lungs were collected from 30 aged donkeys at routine necropsy examination at two UK donkey sanctuaries between June 2009 and September 2012. 19 ‘APF affected’ donkeys had evidence of pulmonary fibrosis on gross examination while 11 ‘control’ animals had grossly normal lungs. Lungs were manually inflated prior to clamping of the trachea and gross images were photographed with a digital camera. 16 whole inflated ex vivo lungs (11 APF, 5 controls) were then imaged with high resolution computed tomography (HRCT). Tissue samples were collected from each lung into 10% buffered formalin according to a standard protocol before undergoing routine processing to paraffin blocks. Sections were routinely stained with haematoxylin and eosin (H&E), elastic van Gieson (EVG) and Masson’s trichrome (MT). HRCT images and histology sections were reviewed independently and blindly by a radiologist and pathologist respectively from both medical and veterinary fields. Sections and HRCT images were categorised as ‘definite’, ‘consistent with’ or ‘inconsistent with’ with regard to PPFE using criteria described by Reddy et al (2012). Cases were categorised as ‘definite’ on either CT or histology if there was pleural thickening with associated subpleural fibrosis either concentrated in upper or dorsal lung lobes (with respect to CT evaluation) or demonstrating intra-alveolar fibrosis with alveolar septal elastosis (with respect to histological evaluation of EVG sections). CT images were categorised as ‘consistent with’ if there was dorsal lobe pleural thickening and associated subpleural fibrosis but the distribution of fibrosis was not concentrated in the dorsal lung lobes or there was evidence of coexistent lung disease elsewhere. Histology sections were categorised as ‘consistent with’ if intra-alveolar fibrosis was present but either not associated with pleural fibrosis, not predominantly subpleural or not in a dorsal lobe biopsy. ‘Inconsistent with’ was assigned to cases that lacked the aforementioned features either on CT or histology.

Results

Ages of ‘APF affected’ (median 31 years, range 14-53) and ‘control’ (median 28 years, range 4-36) donkeys at the time of death were not significantly different (Mann Whitney, p>0.05). The donkeys comprised 11 geldings and 19 entire jennies. 10/19 APF affected cases were euthanased on humane grounds due to respiratory disease, while 9 were euthanased on humane grounds for other reasons and pulmonary fibrosis was an incidental post mortem finding. 10/19 APF affected cases were categorised as either ‘definite’ or ‘consistent with’ PPFE on histological evaluation, while 9 showed histological evidence of pleuroparenchymal fibrosis but this did not have an intra-alveolar distribution. 8/11 APF affected cases were categorised as either ‘definite’ or ‘consistent with’ PPFE on evaluation of HRCT images. Two of the remaining 3 cases showed pleural and subpleural fibrosis concentrated in the ventral lung lobes while one demonstrated diffuse ground glass opacity with minimal pleural fibrosis. Histological evaluation of these three cases also resulted in an ‘inconsistent with’ classification. All control cases were classified as ‘inconsistent with’ on both HRCT and histology.

Conclusions

APF is a common yet rarely diagnosed and apparently untreatable syndrome of aged donkeys. This study is the first to combine HRCT and histological data to characterise and document pathological features of APF. We conclude that the majority of cases of APF share key pathological features with human PPFE. Further study of APF may yield valuable information to help elucidate the aetiopathogenesis of this emerging human disease.

Acknowledgements

The authors gratefully acknowledge funding from the MRC.

References

S. K. Frankel, C. D. Cool, D. A. Lynch, K. K. Brown. 2004, CHEST Journal 126(6).
L. Morrow, K. Smith, R. Piercy et al. 2010, Journal of Comparative Pathology.
T. L. Reddy, M. Tominaga, D. M. Hansell, et al. 2012, European Respiratory Journal 40(2): 377.

Nutrition

A donkey foal should be weaned gradually from 6 months of age and able to graze and eat supplementary straw feed. Barley straw is the forage of choice for healthy donkeys with good dentition, as it is low in calorie content while high in fibre, which aids slow digestion and reduces the risk of gastric ulceration. Straw can also be supplemented with hay in cold weather or if extra energy is required. If excess calories are provided to young donkeys, there is a risk of development of orthopaedic conditions including flexor tendon contractures leading to club foot. To balance the high-fibre diet, a low-calorie vitamin/mineral balancer ration is needed until the foal is at least 2 years old or up to 3 years in the larger breeds of donkey. Top Spec provide a donkey specific forage balancer that is appropriate for young donkeys.

Donkeys have lower nutritional requirements compared with horses. Aim to feed 1.3–1.7% of bodyweight in dry matter, the amount dependent on the weather and the individual animal. The donkey’s body condition score should be measured at least four times a year, while weigh tapes and donkey weight normograms help to monitor for slow, steady weight gain. Weaning the foal can be a stressful time for the jenny, so she needs similar careful management and monitoring.

Castration

Castration is a valuable tool to reduce the population of unwanted donkeys and encourage responsible ownership. The optimum time to castrate a donkey is between 6 and 18 months, although some reports suggest that a jack may be sexually mature by 12 months. Donkeys castrated after 18 months are more likely to retain stallion-like behaviours, and to have complications from surgery due to extensive fat deposits in the scrotum, larger testicles and associated blood vessels. Ensure a thorough preoperative check; many donkeys will have had no veterinary contact until castration. Check for heart murmurs and subclinical lung disease. Discuss vaccination and worming programmes: at a minimum ensure tetanus protection. For most young donkeys, a field castration is adequate. Use a weigh tape or weight estimator to calculate weight. Take a qualified assistant vet or nurse to administer the anaesthetic and top-up doses, as the procedure takes longer than a standing castration. Owners are not suitable assistants. For field anaesthesia, remember to take equipment to protect the donkey’s face and eyes: towels, eye drops, padded head collar, etc.

The Donkey Sanctuary vets prefer to use a standard closed technique for castration of donkeys. The donkey is placed under general anaesthesia, and local anaesthesia is used in the testicle (5–10 mL depending on size). The upper hindlimb is held or roped out of the way. The area may need to be clipped as the scrotum is frequently covered with hair. In the young donkey there should be minimal swelling post-operatively; we use analgesia at donkey doses, for 3–5 days, and depocillin intramuscularly usually for 3 days. Encourage exercise daily; it is useful to cold hose the inguinal area to reduce swelling, avoiding saturating the wound with water. Monitor appetite, faecal output, and demeanour for a week post-surgery. Complications include haemorrhage and infection. If blood is dripping faster than 1 drop/second and not slowing, consider external pressure or re-anaesthetise to locate the source. Infection manifests as a slow-healing wound, discharge, and a painful thickening of the remaining cord tissue. Surgical investigation is often required. In cryptorchid donkeys the anti-Mullerian hormone (AMH) test is proven to detect retained testicular tissue. If this test is not available, a human chorionic gonadotrophin (hCG) stimulation test is required as the oestrone sulfate test is unreliable in donkeys.

Sedation and anaesthesia

Many young donkeys are not well handled, and we must provide good sedation in a welfare-friendly manner. Aim to keep bonded companions together to reduce stress, and consider oral or i.m. sedation if required before attempting i.v. access.

Young colts can have thick coats and a well-developed ventral neck muscle – clip the vein, elevate the head and aim for the jugular above or below the mid third of the neck. A catheter is needed to top up anaesthesia for castrations. Donkey skin is relatively thick so use a scalpel to nick the skin before inserting the catheter; using a bleb of local anaesthetic makes this easier. Typical equine doses of alpha-2 agonists work for donkeys, but be prepared to increase the dose if the donkey is stressed and do not induce anaesthesia until the head has dropped below the withers. Ketamine at a dose of 2.2–3 mg/kg is typically used together with diazepam at 0.1 mg/kg for induction. Multimodal analgesia is provided with the use of an NSAID, an opioid (typically butorphanol) and local analgesia. Donkeys metabolise ketamine faster than horses so be prepared to top up at timed 10-minute intervals with one-third of the induction dose.

If a triple drip is used for anaesthesia, use a recipe appropriate for donkeys, for example: 300 mL saline, 225 mL 10% guaphenesin, 225 mg xylazine and 900 mg ketamine. Avoid doses of guaphenesin above 150 mg/kg (1.5 mL/kg of 10% solution), as this can cause respiratory and cardiovascular depression. Decreased depth of anaesthesia is often preceded by increased rate and depth of respiration before movement occurs; monitor carefully.

Recovery from anaesthesia is usually good in donkeys, unless multiple ketamine top ups have been used. Be prepared to re-sedate with an alpha-2 agonist. A typical 180 kg donkey requires a size 16 mm endotracheal tube, but have a range of sizes between 14 and 18 mm available. Donkeys can be difficult to intubate due to the narrow epiglottis and caudally angled larynx.

Introduction

A 3-year-old castrated male donkey was presented with multifocal, moderately firm, dull, white nodules, varying in size from 0.5-2.0 cm in diameter. The nodules were located on the skin of the penile shaft and showed depigmentation. Other locations on the body were not involved.

Materials and methods

Surgical excision of the nodules was performed. All formalin-fixed samples were embedded in paraffin and routinely processed for histopathological examination.

Results

Histopathological examination revealed presence of multifocal, round to oval, protozoal cysts within the dermis, with a size of 150-500 μm. The mature cyst walls consisted of four distinct layers, including an outer, hyalinized, eosinophilic layer of collagen fibers, a thin homogenous intermediate layer, a layer consisting of the cytoplasm of the host fibroblast with a compressed nucleus, and an inner layer that formed the parasitophorous vacuole. The vacuole was filled with numerous bradyzoites of 2 x 8 μm. There was a mild to moderate, superficial to mid-dermal infiltration of lymphocytes, plasma cells, macrophages and eosinophils, surrounding the cysts and blood vessels. The hair follicles were atrophic. The overlying epidermis showed mild acanthosis and orthokeratotic hyperkeratosis. Based on the histopathological findings, a diagnosis of Besnoitia sp. infection was made.

Conclusions

Besnoitiosis is an emerging disease in cattle in Europe. Few outbreaks have been reported in donkeys in the USA. To our knowledge, this is the first report in the literature of Besnoitia sp. infection in an European donkey.

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Introduction

Many equids are vaccinated against equine influenza annually as it causes a highly contagious respiratory infection. In horses, both age and adiposity contribute to increased levels of inflammatory markers, which could affect the response to vaccination. In humans, a chronic inflammatory state associated with obesity can impair response to vaccination or infection (Sheridan et al., 2012). The objective of this study was to determine which factors would influence the response to the equine influenza vaccination.

Material and methods

Surplus to diagnostic requirement serum samples were obtained from 55 donkeys that had recently received a booster dose of a commercially-available inactivated virus equine influenza vaccine. Antibody levels against a component strain of the vaccine (influenza A/equine/Newmarket/2/93 [H3N8]) were measured using the single radial haemolysis assay. An equine-specific ELISA (R&D Systems) was used to measure serum tumour necrosis factor alpha (TNF-α). High molecular weight (HMW) adiponectin was measured using a human ELISA kit (Millipore) previously shown to be relevant for the horse (Woolridge et al., 2012). Non-esterified fatty acid (NEFA) levels were measured using a kit from Randox with some modifications to the manufacturer’s instructions. Additional data including age, weight, body condition score (BCS), and total cholesterol and triglyceride serum levels were kindly provided by the Donkey Sanctuary. Univariate analysis was conducted using Pearson correlation for normally distributed variables and Spearman’s rank correlation for variables that were not normally distributed. To evaluate the effect of gender, comparisons were made using a t-test for normally-distributed variables or a Mann-Whitney test for variables not normally distributed. Significant differences were determined at the level of p<0.05.

Results

In this study, the factor with the greatest influence on the response to vaccination was gender, with females having significantly higher antibody levels than males (Table 1). In addition, the mean age of the female donkeys was significantly greater than that of the male donkeys. There was no correlation between BCS or weight and antibody levels, but NEFA levels were negatively correlated with antibody levels (p=0.044). Associations between NEFA and age, weight and days since vaccination also reached statistical significance. Triglyceride levels were also positively correlated with days since vaccination although antibody levels were not. Positive correlations were seen between serum adiponectin and age, and triglyceride and cholesterol levels.

Table 1. Influence of gender on variables measured

Variable Mean ±SD (Range) p-value
Males (n=28) Females (n=27)
Age (years) 19.2 ±11.8 25.0 ±6.4 .028
Antibody (area of lysis - mm2) 178.9 ±39.3 208.9 ±42.3 .009

Conclusions

Negative correlations were expected between antibody levels and both age and BCS, but were not seen. This may have been confounded by the narrow distribution of BCS in the study population (the majority had a BCS score of 2.5–3.5 on a scale of 1–5) and the overwhelming influence of gender with female donkeys having a higher antibody response despite a greater mean age. It has been demonstrated in human subjects that influenza vaccination can cause alterations to the lipid profile (Tsai et al., 2005). The correlation between NEFA and serum antibody levels warrants further investigation as does the finding that gender has a significant impact on response to equine influenza vaccination in donkeys.

Acknowledgments The authors gratefully acknowledge The Donkey Sanctuary for providing surplus to diagnostic requirement serum samples and data, Waltham Centre for Pet Nutrition for funding the study and Dr Marnie Brennan for assistance with the statistical analysis.

References

P. A. Sheridan, H. A. Paich, J. Handy, E. A. Karlsson, M. G. Hudgens, A. B. Sammon, L. A. Holland, S. Weir, T. L. Noah, M. A. Beck. 2012. Obesity is associated with impaired immune response to influenza vaccination in humans. International Journal of Obesity, 36, 1072-1077.

M. Y. Tsai, N. Q. Hanson, R. J. Straka, T. R. Hoke, J. M. Ordovas, J. M. Peacock, V. L. Arends, D. K. Arnett. 2005. Effect of influenza vaccine on markers of inflammation and lipid profile. The Journal of Laboratory and Clinical Medicine, 145, 323-327.

A. A. Wooldridge, H. G. Edwards, E. P. Plaisance, R. Applegate, D. R. Taylor, J. Taintor, Q. Zhong, R. L. Judd. 2012. Evaluation of high-molecular weight adiponectin in horses. American Journal of Veterinary Research, 73, 1230-40.

In the last one hundred years, tractors with much more power replaced animals all over the world, except in developing countries. Besides the advantage in power, the use of tractors increases agriculture productivity and time efficiency of field operations. Despite the unquestionable advantage of tractors for agriculture performance, in recent years the interest by animal traction is growing, even in developed countries. It is important to understand why. This interest usually is linked to small scale farming, but not only. Environmental concern is one of the most significant reasons, in different aspects: use of working animals as a renewable source of energy; forest management in protected areas to protect soil physical properties related to compaction affected by tillage treatments; field operations in environment protected areas, among others. In old vineyards, but producing a high value output, plant spacing turn animal traction without alternative. The maintenance of the gene pool in domestic animals; the creation of jobs in rural areas, such as farrier, harness maker and implement manufacturer, are also important issues. New challenges: new implements must be designed and new mechanical solutions achieved.

The hindgut microbiota of equines enables them to utilize forage/grazing based diets which contain a substantial proportion of lignocellulosic fibres. These fibres are a structural barrier that gut bacteria need to overcome when accessing plant nutrients, as well as being a challenging and structurally complex substrate that can be utilized. The limited dietary energy available from these ‘natural’ diets however means that many equines are supplemented with energy-dense concentrate feeds in order to fulfil their dietary energy requirements. Use of energy-dense concentrate feeds however can change the equine hindgut microbiome, and lead to the development of gut-mediated diseases (i.e. fermentative acidosis, laminitis, colic and stomach ulcers). There is therefore a clear need to optimize the utilization of lignocellulosic fibres in the equine hindgut in order to minimize the need for dietary supplementation. The most effective of the fibre-degrading gut microbes, anaerobic fungi (phylum Neocallimastigomycota), are known to be a normal member of the equine gut microbiota. Despite this however, they have been largely overlooked in equine gut microbiology studies to date. Research being conducted within the EU funded EQUIANFUN project will therefore establish baseline knowledge of the phylogeny, community structure, physiology and nutritional impact of anaerobic fungi in the equine hindgut. The insights gained will inform the development of novel strategies to promote indigenous anaerobic fungal communities in the equine hindgut, enabling optimization of the use of dietary forage as an energy source in equids. Reduction of the use of energy–dense diets and applying targeted nutritional strategies for optimizing microbial health may counteract processes in the gastrointestinal tract that have been associated with disease. Anaerobic fungi therefore offer the potential to enable significant advances to be made in the optimisation of the nutrition, health and welfare of all domesticated equids.