This study presents the first insights into the genetic diversity and structure of the American donkey metapopulation. The primary objectives were to detect the main structural features underlying variability among American donkey populations, identify boundaries between differentiated gene pools, and draw the main colonization pathways since the introduction of donkeys into America in the 15th century. A panel of 14 microsatellite markers was applied for genotyping 350 American donkeys from 13 countries. The genetic structure of this metapopulation was analysed using descriptive statistics and Bayesian model-based methods. These populations were then compared to a database containing information on 476 individuals from 11 European breeds to identify the most likely ancestral donor populations. Results showed the presence of two distinct genetic pools, with confluence of the two in Colombia. The southern pool showed a unique genetic signature subsequent to an older founder event, but lacked any significant influence of modern gene flow from Europe. The northern pool, conversely, may have retained more ancestral polymorphisms and/or have experienced modern gene flow from Spanish breeds. The Andalusian and, to a lesser extent, the Catalan breeds have left a more pronounced footprint in some of the American donkey populations analysed.

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Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3′)-IIIa, ant (4′)-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3′)-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.

To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichiacoli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

In Portugal, equine parasitism in pasture animals is characterized by high parasitic burden and high helminthic biodiversity; both factors are potentially pathogenic for their hosts. The decrease in the number of donkeys over the last years in Portugal, their importance in rural lowland and mountain ecosystems and pastures and the scarce information regarding their parasitism led to this research, which aimed to evaluate the parasitological status of a Miranda donkey breed population, a native breed mainly located in the northeast of Portugal. This study provides better knowledge of their gastrointestinal parasitism, particularly strongyles, and the assessment of a targeted selective treatment (TST) as an alternative control approach of their parasitism. A longitudinal observational study was developed during a period of five years in a population of 62 Miranda donkeys. At first, strategic deworming of these animals was performed every semester, but this was progressively replaced by a TST approach according to the levels of Eggs per Gram (EPG). This new deworming strategy was conducted in association with a regular parasitological monitoring of the animals every three months, being dewormed with ivermectin when egg shedding was higher than 500 EPG. Over the study period, a decrease of the annual prevalence rate of infection by gastrointestinal strongyles was observed, from 35.5% to 19.4%, as well as a negative binomial distribution of parasitic strongyles in donkeys submitted to selective anthelminthic control. The prevalence rate of infection was higher in females (39.5%), in individuals under four years (46.7%) and in those presenting a lower body condition (40.8%). The egg output was higher in animals younger than four years (589.3 EPG) than in those older than 10 years (533.6 EPG) (p < 0.05). However, no differences were observed according to sex during the study period. Results from this study allowed to note the influence of swampy pastures and of the weather changes in the epidemiology of strongylosis in Miranda donkeys. Moreover, it was possible to establish the annual epidemiological curve of strongyle egg shedding, with June being the month with the highest EPG, December having the lowest EPG and March and September showing intermediate numbers. Overall, a lower biodiversity of gastrointestinal parasites was observed. Cyathostomum sensu lato was the most prevalent genus and Strongylus vulgaris was the most observed large strongyle of the Strongylidae family. Trichostrongylus axei and Parascaris sp. were other nematodes with a minor frequency. The higher prevalence of strongyles at the beginning of the study showed a progressive decrease throughout the research period, and also for parasite biodiversity. Therefore, a targeted selective treatment seems to be a rational anthelminthic control approach in Miranda donkey strongyle infection and in other gastrointestinal parasites, since it reduces the antiparasitic treatments, the parasite’ prevalence and the EPG level. However, a loss of parasite biodiversity was noted at the end of the study period, as Cyathostominae were the only isolated strongyles. This can be a challenging situation in the long run, taking the ability of these nematodes to adapt easily to any deworming program, meaning that fecal EPG monitoring should be kept as a rule to a rational parasite control program.

Toxoplasma gondii and Leishmania infantum are zoonotic protozoal parasites. Serum samples were obtained from 186 donkeys (Equus africanus asinus) from Portugal and assessed for antibodies to T. gondii by the modified agglutination test (MAT). For titration of antibodies to Leishmania spp. the direct agglutination test was used (DAT). Eleven donkeys were seropositive for T. gondii with titres of 20 (n = 7), 80 (n = 2), 640 (n = 1) and ≥ 2560 (n = 1). One donkey was seropositive for Leishmania spp. (titre of 800). Donkeys in Portugal are exposed to and can be infected with T. gondii and Leishmania spp.

Due to the long courtship needed to attain excitation and erection, donkey semen collection can take up to 90 min. ProstaglandinF2α (PGF2α) has been reported to hasten the onset of erection and ejaculation in domesticated mammals, presumably by inducing smooth muscle contractions in the internal genitalia. However, while it has been anecdotally used in donkeys, it has yet to be critically evaluated. This study aimed to compare behavioral and semen parameters in Catalan, Balearic, Amiata, and Miranda jacks treated with the PGF2α analogue cloprostenol sodium immediately prior to exposure to an estrus jenny. Nineteen donkeys were assigned in a crossover design to receive cloprostenol sodium (125 µg, i.m.; n = 53 collections) or saline (1 mL, i.m.; n = 53 collections). There were no differences for erection (52/53 vs. 52/53) or ejaculation (52/53 vs. 48/53) for collection attempts assigned to saline or cloprostenol sodium, respectively. Cloprostenol sodium significantly hastened treatment-to-erection and treatment-to-ejaculation times from 12.0 ± 1.6 to 6.0 ± 1.6 min and from 14.0 ± 1.4 to 9.6 ± 1.4 min, respectively. Significant effects of breed and age were observed in behavioral and parameters, but there were no effects of cloprostenol sodium administration on semen parameters. In conclusion, cloprostenol sodium administration immediately prior to semen collection hastened time to collect semen in donkeys with no detrimental effects on semen quality and can be used by practitioners to circumvent long delays in donkey semen collection.

The increase in donkeys treated by practitioners in recent years has led to an increased interest in finding more information on basic biochemical preliminary reference values. The aims of this study were to measure Se and Vit E levels in plasma from Miranda jennies peripartum and postpartum and in their foals to compare blood profiles of the jenny and foal related to the overall foal’s health. Twenty-two healthy peripartum and postpartum Miranda donkeys were sampled (12 jennies and 10 foals) in the northeast of Portugal (Atenor and Paradela) from May to November, 2018. Amounts of selenium in soil were significantly correlated (0.97) to concentrations of selenium in jennies (42.412 μg/L in Atenor and 9.612 μg/L in Paradela) and foals (19.378 μg/L in Atenor and 6.430 μg/L in Paradela). Selenium levels were lower in foals than adults and in males than females. Vitamin E was associated with overall foal health. Foals with a mean vitamin E of 3.585–5.307 mg/L showed signs of weakness, but carpal flexural deformities were observed when the average vitamin E was 11.520 mg/L. Low vitamin E levels (5.307 mg/L) in jennies were related to foal mortality. Diets, location, parity, and age affect blood profiles of jennies and, ultimately, foal health.