donkey

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 Ethiopia the Donkey Sanctuary started its health and welfare activities in 1994. This was the time when the International Donkey Protection Trust and Addis Ababa University concluded an agreement and signed a memorandum of understanding. Since that time and up to 2003, activities were centred in the districts of Bereh, Boset, Ada and Akaki and Addis Ababa Grain Market located at a radius of 100-150 kms from Debre Zeit, the centre with a stationery clinic. The facilities of the latter include an open-air clinic, surgical theatre, laboratory, store for drugs, boxes for mules and donkeys, offices and a training room. As of 2004, three districts namely: Dugda Bora, Lume and Sebata have been added as sites of operation. Resistance at the very beginning by donkey owners to bring their animals for treatment was a problem but over the years the service has obtained wide acceptance, the trend exhibiting exponential growth: Ada (R2=0.87), Akaki (R2=0.88), Bereh (R2=0.70), Boset (R2=0.70), and for mules (R2=0.84). The clinic has so far provided treatments to 289,999 donkeys and 14,665 mules, and 217,609 owners have benefited. In 2003 two satellite projects have been set up in the northern part of Ethiopia: Amhara and Tigray and have so far treated more than 72,000 donkeys and mules and more than 29,000 owners have benefited. The centre at Debre Zeit was also designed to cater for local and international training, to date it has provided training to 217 veterinary professionals (28 foreign vets, 41 local vets, 17 foreign and 101 local animal health assistants, 30 local animal health technicians) in donkey medicine, surgery, farriery and general welfare. Since 2002, 297 clinical year students enrolled for the DVM degree in the Faculty of Veterinary Medicine of Addis Ababa University have been trained in donkey medicine and surgery. Three to four clinical year students accompany mobile teams in field operations on a weekly rotation basis. Recently, the project has embarked on an education and extension programme in conjunction with its treatment activities and so far 51,513 owners have benefited. In the recently launched school education programme 1014 students in 7 primary schools are attending sessions in the basics of animal welfare. Baseline data development for the understanding of the health and welfare problems of donkeys in Ethiopia is being pursued by the project staff and through the supervision of DVM thesis involving final year students.

Pain due lesions and clinical conditions is one of the main welfare concerns of the more than 42 millions donkeys that presently exist in the world. Yet, the knowledge to gauge pain in donkeys is lacking, misunderstood and/or not validated (Ashley, 2005).

Pain (yes/no/uncertain) and its severity (VAS; no pain=0 to worst pain=100mm) was assessed in 403 donkeys’ ante-mortem (ATM) and post-mortem (PTM). Also behaviours/signs (BS) and pain related lesions (PRL) were assessed ATM and PTM, respectively. Using principal component analysis the more than 53 BS and 238 PRL observed were narrowed to 58 biologically meaningful component or groups (14 BS and 44 PRL components, respectively). Components were used as risk factors in multiple regression analysis to identify which BS and/or PRL are commonly used in clinician’s (veterinary/pathologist) decision making process to determine whether a donkey ‘is’ (i.e. ATM) or ‘was’ (i.e. PTM) in pain and its severity (mild to severe). Furthermore, multiple correlations were made to understand which BS relate significantly with specific PRL and how.

A cross tabulation between pain ATM and PTM, where pain related lesions are used as a quasi-gold standard of pain assessment; identify that 2 in 10 donkeys are wrongly assumed as in NO-PAIN. Moreover, only 43% of the donkey observations are used by clinicians to make their opinion on donkey pain and its severity (i.e. 7 BS and 18 PRL components were significantly associated with pain as stated by clinicians). Yet, multiple correlations showed 20 plausible biologically meaningful relationships between BS and PRL; some currently not used by clinicians.

This methodology, previously successfully used in humans (Gregory, 2010) is novel to donkey veterinary medicine and warrants further research to consolidate findings. Nonetheless, the achieved correlation list of behaviours vs. pathologies is a significant work with valid applications in donkey pain identification and prognosis.

References

F. H. Ashley, A. E. Waterman-Pearson, H. R. Whay (2005) Behavioural assessment of pain in horses and donkeys: application to clinical practice and future studies, Equine Vet Journal, 37(6), 565-575.

N. G. Gregory (2010) Relationships between pathology and pain severities: a review. Animal Welfare 19, 437-448.

This paper proposes an approach to use pain-relevant pathologies to enhance our understanding of the clinical and behavioural signs of pain in donkeys and outlines initial results of this ongoing investigation.

The methodology is summarized as follows. Trained veterinary clinicians examined live donkeys under two situations: A) before being euthanized due to a terminal illness or reduced quality of life (n= 347 sedentary donkeys in UK; DU) or B) when about to be slaughtered in an abattoir (n=164 working donkeys in Mexico; DM). The animals that represent populations in terms of age (years) for DU and DM respectively, were: <5 = 0.5%, 31.7%; 5-15 = 4%; 67.7%; 16-20 = 6%, 0.6%; >20 = 89.5% and 0%. For sex were: stallions 0.5%, 44%; geldings 52.2%, 8%; females 47.3%, 48% for DU and DM respectively. The body condition was: <2 =18.5%, 44.7%; 2.5 – 3 = 56.5%, 54%; >3.5 = 25%, 1% for DU and DM respectively and the girth was: 115 cm ±SD 9.6, 112cm ±SD 7.3 for DU and DM respectively.

The clinical examination (CE) included the oral mucosa, heart & respiratory rate, rectal temperature, plus an evaluation of 6 demeanours and 47 behaviours/signs that could relate to pain. At this point an overall pain visual analogue score was derived (VAS 0cm = no pain to 10cm = the worst pain). At post-mortem (PM), lesions/pathologies were noted, grouped by system-organ/tissue, ranked (mild, moderate, severe) and classified according to the following potentially painful pathologies: 1) trauma, 2) inflammation, 3) over-distension 4) perforation/ rupture, 5) stripping/ ulceration, 6) adhesions, 7) swelling, 8) exposure of sub-chondral bone. From these observations a second VAS was produced.

Raw correlations from the two populations showed that donkeys given a higher VAS at CE and PM presented with a greater severity of lesions in more systems as well as a higher heart rate at CE than those donkeys with a lower PM VAS. Moderate to severe pain identified CE was often recognized as severe pain at the PM stage. These initial observations show promise, and so further analysis will be done to test the relationships between pain indicators and pain pathologies.