Post fetal death development of endometrial cups in a Jenny donkey (Equus asinus)

Serial ultrasound examinations of the gravid uterus of a Jenny donkey (Equus asinus) carrying a transferred extraspecies horse (Equus caballus) conceptus between 11 and 33 days of gestation demonstrated that the embryo died before Day 33 even though the conceptus persisted in the uterus and the horse chorionic girdle invaded the maternal endometrium to form a circle of typically large horse endometrial cups in the right uterine horn. The high concentrations of horse CG secreted by these cups with its higher than usual ratio of FSH-like to LH-like biological activities of 1.4 compared to the 0.1 ratio of normal donkey CG hyper-stimulated the donkey’s ovaries to develop multiple large accessory follicles and resulting secondary corpora lutea which secreted high concentrations of progestagens that persisted in her bloodstream for the next 90 days. This case illustrated that, in all likelihood, girdle invasion can happen after the death of the embryo indicating autonomy of the chorionic girdle.     

Successful Non-Surgical Transfer of Horse Embryos to Mule Recipients

Mules, hybrids resulting from the mating of a horse mare (Equus caballus, 2n = 64) to a Jack donkey (E. asinus, 2n = 62), are generally infertile. Five horse embryos were transferred non‐surgically to two cyclic and one acyclic recipient mules. In the mares and cycling mules, oestrus and ovulation were induced with, respectively, d ‐cloprostenol and human chorionic gonadotrophin (hCG). The acyclic mule, on the other hand, received oestradiol benzoate when the embryo donor was showing oestrus and progesterone after the donor had ovulated and until pregnancy diagnosis. Non‐surgical embryo collections were attempted on day 7 after ovulation and recovered embryos were transferred transcervically into the mules’ uteri. Mules that became pregnant were blood sampled serially for equine chorion gonadotrophin (eCG), progestagen and total conjugated oestrogen concentrations until around 6 months of gestation. The three embryos transferred to the acyclic mule did not produce any pregnancies whereas both embryos transferred to the cycling mules resulted in the birth of live foals. The peak concentration and duration of secretion of eCG differed markedly between the two pregnant mules, although both animals appeared to develop secondary corpora lutea beyond day 40 of gestation, as in normal intraspecies horse pregnancy. Moreover, the rise in serum oestrogen concentrations from around day 90 was also similar to that seen in normal pregnant mares. Parturition occurred spontaneously on day 348 of gestation in both mules and the resulting colt foals developed normally to weaning. Thus, cycling mules can carry a horse conceptus after non‐surgical embryo transfer and give birth to a normal mature foal.     

Degenerative myelopathy in two equids

Degenerative myelopathy was diagnosed in a donkey Equus asinus and a Welsh pony Equus caballus. Both equids were presented for gradually progressive incoordination. Neurologic examination revealed symmetrical weakness and ataxia which was much more prominent in the rear limbs than in the front limbs. Laboratory tests including a complete blood count, serum biochemistry, cerebrospinal fluid analysis and lateral cervical radiographs were performed in both equids and were unremarkable. Both equids were euthanized and microscopic examination of the spinal cords revealed diffuse neuronal degeneration, demyelination and neuraxonal dystrophy.     

Comparative endocrinological responses to short transportation of Equidae (Equus asinus and Equus caballus)

In order to evaluate the effects of short transportation on β‐endorphin, adrenocorticotropic hormone (ACTH) and cortisol changes, 12 healthy stallions of Equidae (Equus asinus and Equus caballus) were studied before and after transportation of 50 km. Blood samples were collected 1 week before transportation in basal conditions, immediately before loading and after transportation and unloading, on their arrival at the breeding station. Compared to basal and before values, donkeys showed an increase in circulating ACTH (P < 0.001) and cortisol (P < 0.0005) levels after transportation and higher ACTH (P < 0.01) levels than horses after transportation. A positive and significant correlation (r = 0.885; P < 0.01) between ACTH and cortisol levels after transportation was found. No significant differences were observed for β‐endorphin levels. Compared to basal and before values, horses showed higher cortisol (P < 0.005) levels after transportation and no significant differences were observed for ACTH and β‐endorphin levels in donkeys. Horses facing forward (direction of travel) showed higher (P < 0.01) β‐endorphin levels after transportation than donkeys; horses facing backward (the opposite direction of travel) showed lower (P < 0.05) ACTH levels after transportation. The results indicate that short transportation induces a preferential activation of the hypothalamus‐pituitary‐axis (HPA), with significant release of ACTH and cortisol in donkeys and only of cortisol in horses, suggesting that transportation for donkeys may be more stressful than horses.     

Possible morphological differentiation of horse and mule kidneys from those of donkey and hinny

By means of plastic injections it was possible to determine that the kidneys of horse and mule have terminal recesses while those of the donkey and hinny have no terminal recesses.     

Multifocal Coccidioides immitis in a donkey (Equus asinus)

This case report discusses the clinical signs, diagnostic findings, treatment and outcome for a donkey (Equus asinus) diagnosed with coccidioidomycosis. At presentation, the donkey was lame, was in poor body condition, had a draining tract over the left femur and was hyporexic. Further investigation of the draining tract via radiography and ultrasonography showed a focal lesion of extensive bony proliferation and lysis on the mid-shaft of the left femur. There was also a soft tissue swelling over the fifth left rib. AGID titres were positive for Coccidioides immitis. Recommended treatment for C. immitis consists of two antifungal classes, the azoles and amphotericin B, with this donkey being prescribed daily fluconazole. A recheck 3 months from initial diagnosis showed marked improvement of clinical signs, but the C. immitis titre remained unchanged. The 18-month recheck showed improvement radiographically and significantly decreased titres. After 3 years of daily treatment of fluconazole, the C. immitis titre was decreased, but radiographs of the femur still showed persistent bony proliferation.     

Mitochondrial DNA variation and genetic relationships in Spanish donkey breeds (Equus asinus)

In this study, the mitochondrial DNA diversity of six Spanish donkey breeds and two African donkey populations (one from Morocco and the other from Zimbabwe) was analysed. A total of 79 animals were sequenced for 313 bp of the cytochrome b gene, and 91 individuals for 383 bp of the D‐loop region or control‐region. Sequence comparisons and phylogenetic analyses of both Spanish and African populations revealed low diversity. Only six and seven haplotypes respectively were found in cytochrome b and the D‐loop region. Relatively low nucleotide diversity (π) values were detected in these populations. The π values, from the D‐loop region, ranged from 0.0006 to 0.0169 for the Catalana and Andaluza breeds, respectively. The obtained results seem to confirm the existence of two divergent maternal lineages of African origin (Equus asinus africanus and E. a. somaliensis). In this paper the genetic relationships between these breeds are analysed and compared with those obtained in other European populations. Also, the data on the genetic relationships between the populations, obtained from nuclear DNA (microsatellites) and mitochondrial DNA (cytochrome b and D‐loop region) is argued and interpreted.     

A centric fission and heterochromatin polymorphism in Equus asinus Spanish breeds

In this work a chromosomal study in Equus asinus is presented. Forty‐two specimens, belonging to five different Spanish breeds in risk of disappearing, have been analysed. Metaphases have been sequentially stained (uniform, G‐banded and C‐banded) and analysed. All specimens, except one, presented 2n = 62 chromosomes. Variation in chromosome number (2n = 63) is because of a fission in one chromosome of pair no. 3, resulting in two acrocentric chromosomes. Polymorphisms affecting the size of heterochromatic C+ bands and the presence of hetrochromatic 1p and 4p arms are also analysed. The results show no polymorphisms which can be attributed to a particular breed. From the chromosomal point of view, all Spanish asinine specimens correspond to a single population. Rearrangements detected suggest a fission tendency in the homologous E. asinus no. 3 chromosome in Equidae species.     

Molecular and cytogenetic paternity testing of a male offspring of a hinny

An alleged male foal of a female mule, whose sire and grandparents were unknown, was identified for its pedigree. Parentage testing was conducted by comparing polymorphism of 12 microsatellite DNA sites and mitochondrial D‐loop sequences of the male foal and the female mule. Both the sequence analysis of species‐specific DNA fragments and a cytogenetic analysis were performed to identify the species of the foal and its parents. The results showed that the alleged female mule is actually a hinny, and the male foal, which possesses 62 chromosomes, qualifies as an offspring of the female hinny and a jack donkey.     

Cloning,tissue distribution and functional characterization of the donkey (Equus asinus) oligopeptide transporter 2

Oligopeptide transporter 2 (PepT2) is an important transporter of oligopeptides. In the present study, we describe the molecular cloning, tissue distribution and functional characterization of a donkey (Equus asinus) PepT2. The cloned cDNA sequence was 2202 bp at full length, encoding a 733 amino acid peptide with a molecular weight of 81.9 kDa and a theoretical pI of 8.92. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of PepT2. The expression of PepT2 in the kidney and lung was significantly higher than that observed in the ileum, duodenum, jejunum, spleen, liver, heart and stomach. Functional characterization by heterologous expression in Chinese hamster ovary cells showed that the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA) by donkey PepT2-Chinese hamster ovary cells was dependent on time, pH and substrate concentration, with a low Km value of 91.51 ± 14.14 μM and a maximum velocity of 41.37 ± 2.193 pmol/min/mg protein. In the present study, for the first time, the expression and functional characteristics of donkey PepT2 were evaluated, the results of which provide new insights and a better understanding of its crucial role in oligopeptide transport in donkeys.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

Seropositivity of Toxoplasma gondii in domestic donkeys (Equus asinus) and isolation of T. gondii from farm cats

Donkeys (Equus asinus) are used as both companion and working animals throughout the world and in some countries, their meat and milk are used for human consumption. Here we report the first serological survey of Toxoplasma gondii in donkeys in the United States. Serum samples from 373 donkeys from eight farms in five states were tested for T. gondii antibodies by the modified agglutination test (MAT). Twenty-four of 373 (6.4%) of donkeys were seropositive, with MAT titers ranging from 25 to ≥200. All seropositive donkeys were Miniature breed. Seropositivity prevalence was 7.0% in female donkeys (20/282) and 4.1% in male donkeys (4/91). No donkeys less than 24 months of age (129) were seropositive, suggesting postnatal transmission of infection. Domestic cats were present on six of the eight farms. Three cats from one farm had MAT titers of 200. Viable T. gondii was isolated from the hearts of two cats, but not from brain tissues. Genotyping of isolate DNA extracted from culture-derived tachyzoites using 10 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico loci) revealed that both isolates were clonal Type II (ToxoDB PCR-RFLP genotype #1). This is the first serological survey for T. gondii in donkeys in the United States, and suggests that donkey milk and meat should be considered as a potential source for human infection. The role of barn cats in the transmission of T. gondii to donkeys on farms warrents further investigation.     

Reference intervals for biochemical and haematological parameters in mature domestic donkeys (Equus asinus) in the UK

Reference intervals (RIs) for haematology and serum biochemistry for donkeys in a temperate climate have been previously published using blood sample results from the resident population of a large donkey shelter in the UK. Periodic review of reference intervals is recommended to ensure their applicability to the patient population and changes in laboratory methods and technologies. The current study aimed to revise the previously published haematology and serum biochemistry values for the adult domestic donkey (Equus asinus) in the UK in the light of a change in analytical equipment at the Donkey Sanctuary laboratory, but also to refine the demography of the sample population with respect to age, physiology and clinical history. Clinical pathology results from 138 clinically healthy mature (4–24 years inclusive) female and castrated male donkeys selected from the resident population of the Donkey Sanctuary, were analysed retrospectively. The animals were blood sampled during the period February 2008 to June 2011 as part of a routine health screen prior to rehoming. Results for a total of 38 biochemical and haematological parameters were analysed including 3 previously unreferenced parameters in addition to those assessed in the previous study. The new reference intervals and median values show very poor transferability with recently derived reference intervals for non‐Thoroughbred horses and only limited transferability with reference intervals previously published for donkeys in the UK. Of particular note is a marked reduction in the upper reference limit for triglycerides of 2.8 mmol/l (from 4.3 mmol/l) since this parameter is used to decide when donkeys are at risk of developing hyperlipaemia. This study demonstrates the value of intermittent review of reference intervals and refinement of study populations. Notwithstanding the caution with which reference interval data from different laboratories should be compared, the lack of transferability of results between donkeys and horses highlights the importance of use of species‐appropriate reference intervals for clinical decision‐making.     

Differentiation of Twelve Actinobacillus pleuropneumoniae Serotypes by Outer Membrane Lipoprotein Gene‐based Restriction Fragment Length Polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)‐amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950‐base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR‐based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR‐based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.     

Identification of tamaraw (Bubalus mindorensis) from natural habitat‐derived fecal samples by PCR‐RFLP analysis of cytochrome b gene

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit‐Baco National Park and species‐known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species‐unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus‐ and tamaraw‐specific primers for PCR of cytochrome b gene were newly designed. The Bubalus‐specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw‐specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR‐RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR‐RFLP analysis also showed that the species‐unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.     

Breed and species comparison of amino acid transport variation in equine erythrocytes

The amino acid permeability of red blood cells from Equus caballus (thoroughbred, Arab, shire and pony), E przewalskii (Przewalski's horse), E asinus (donkey and mule) and E burchelli (common or plains zebra) was measured. Individual animals exhibited stable but widely differing rates of L-[U-14C]alanine uptake in the range 5 to 1554 mumol (litre cells)-1 h-1 (0.2 mM extracellular L-alanine, 37 degrees C). Of the thoroughbreds tested, 30 per cent had red blood cells which were essentially impermeable to L-alanine (5 to 10 mumol (litre cells)-1 h-1, giving transport rates similar to those found previously in amino acid transport-deficient sheep erythrocytes. In contrast, only 3 per cent of the ponies tested had red blood cells impermeable to L-alanine. No cases of erythrocyte amino acid transport deficiency were found in the other horse breeds and species tested.     

First case of sterility associated with sex chromosomal abnormalities in a jenny

Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two‐and‐a‐half‐year‐old jenny characterized by morphological abnormalities of the reproductive tract was cytogenetically analysed using conventional and fluorescent techniques and a group of microsatellite markers (short tandem repeat, STR). At the same time, five ultrasound measures of the reproductive tract were taken and compared with eight contemporary jennies of the same breed. After slaughter, morphological examinations showed that the case study had a blind vaginal vestibule defining an empty pouch that covered the entrance of the cervical os. Histopathological studies demonstrated that this abnormal structure was compatible with a remnant hymen. Molecular markers, STR and fluorescent in situ hybridization determinations revealed that the animal was a 62, XX/61,X mosaic and, therefore, the first case of chromosomal abnormalities in the sex pair reported in donkeys.     

羧化胶乳凝集抑制试验快速鉴别牛肉和马肉的研究

以羧化聚苯乙烯胶乳作为载体,将牛血清和马血清分别共价交联到载体微球的表面,制成免疫微球.将此免疫微球同相应的抗血清配对,进行胶乳凝集抑制试验以鉴别牛肉和马肉.通过对265头份牛肉、242头份马肉、100头份猪肉、60头份羊肉、30头份狗肉、13头份骡肉和4头份驴肉鉴别,牛肉胶乳试剂和马肉胶乳试剂的敏感性均为100%,牛肉胶乳试剂的特异性为100%,马肉胶乳试剂同骡肉和驴肉呈完全交叉反应.实验具有良好的重复性和稳定性,试剂易于保存,本试验可在5min内报告结果,操作简便,适于基层动检部门应用.     

Damage by the bit to the equine interdental space and second lower premolar

Equus caballus mandibles were surveyed in 4 museum collections and 66 domestic horse mandibles were compared with 12 feral and Przewalski mandibles. Periostitis (bone spur formation) of the interdental space (bars of the mouth) was found in not less than 62% of the domestic hemimandibles. Erosion of enamel and dentine was found in 61% of the second lower premolars (Triadan 306 or 406). Eighty‐eight percent of the domestic mandibles showed one or both lesions. The more severe the interdental periostitis, the more likely it was that the 06s were eroded. Twelve feral and Przewalski mandibles were free of both lesions. A clinical examination protocol is described for the identification of bit damage in the live horse. By recommending removal of the bit when damage is found, practitioners can test the author's prediction that the bit is a cause of certain idiopathic diseases and many behavioural problems.