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Aims: We aim to detect beta-lactamase-producing Citrobacter sedlakii from horses and compare the genomic characteristics with isolates from humans., Methods and Result: We characterized phenotypically and genotypically nine C. sedlakii isolates from the feces of horses and then compared them to human-derived isolates using whole genome sequencing and phylogenomic methods. Seven isolates (7/9) were ampicillin-resistant, while at least one isolate was resistant to ceftriaxone, gentamicin, meropenem, and streptomycin. All nine isolates were carriers of the chromosomal-mediated blaSED-1  beta-lactamase gene, which confers resistance to ampicillin. One isolate was positive for the mcr-9 gene that confers resistance to colistin, and another isolate had the aac(6')-lid gene that confers resistance to aminoglycosides. Seven isolates (7/9) were carriers of genes that confer metal resistance to copper, silver, and arsenic. Phylogenetically, two horse-derived isolates clustered together with two human-derived isolates from the NDARO database., Conclusion: The results from our study provide insight into the antimicrobial susceptibility of C. sedlakii in horses, which was previously lacking, and the specific beta-lactamase gene mediating resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Objectives: We performed a retrospective analysis of MRSA isolates collected at the university equine clinic including clinical isolates from 2008 to 2021 and screening environmental, equine and personnel isolates from 2016., Methods: Screening and clinical samples were cultured on Brilliance MRSA 2 and Columbia agar (Oxoid), respectively, with enrichment for environmental samples. Antimicrobial susceptibility was assessed by disc diffusion. All the isolates were characterized by spa typing. Eighteen selected isolates were subjected to WGS with subsequent wgMLST clonal analysis., Results: Among 75 MRSA isolates, five spa types were identified, the majority (n = 67; 89.33%) was t011. All isolates were resistant to cefoxitin and ampicillin and carried the mecA gene. In addition, the isolates were resistant to tetracycline (n = 74; 98.67%), gentamicin (n = 70; 93.33%), enrofloxacin (n = 54; 72.00%), sulfamethoxazole-trimethoprim (n = 5; 6.67%) and lincomycin (n = 3; 4.00%) with corresponding genetic markers for the resistance detected in the sequenced isolates. All 18 sequenced isolates belonged to ST398, 16 carried SCCmec type IVa and two carried SCCmec type Vc (5C2&5). Further, isolates carried aur, hlgA, hlgB and hlgC virulence genes, and five isolates carried sak and scn genes, which are part of the immune evasion cluster. Close genetic relatedness was found between isolates from the staff of the clinic and clinical samples of horses., Conclusions: Repeated introduction and long-term persistence of the equine LA-MRSA subclone (ST398-MRSA-IVa/Vc(5C2&5), t011) among the infected horses at the equine clinic with the colonization of personnel, and the environment contamination that might contribute to transmission were observed., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Crib-biting is a stereotyped oral behavior with poorly understood etiology and pathophysiology. The relationship between the gut microbiome and brain function has been described in behavioral disorders such as schizophrenia, depression and anxiety in humans. In horses, studies of behavioral problems and the microbiome are very limited. This study aimed to characterize the fecal microbiome and the predicted functional profile of horses with and without aerophagia. Fecal samples were collected from 12 Colombian Creole Horses of both sexes, divided into two groups: group 1, composed of six horses with crib-biting (3 females and 3 males), average body weight of 330 ± 10 kg, age of 7.0 ± 1.2 years and body condition score (BCS) of 5/9 ± 1 and group 2, consisting of six horses without crib-biting (3 females and 3 males), average body weight of 335 ± 5 kg, age 6.5 ± 1 years and BCS of 6/9 ± 1. From each horse in both groups fecal total DNA was obtained and 16S ribosomal RNA gene amplicons were sequenced to characterize the bacterial community structure. Community structure and differential abundance analyses revealed significant differences between the two conditions (p < 0.05). Specifically, the fecal microbiota at the family level in crib-biting horses, showing a decrease in Bacteroidales and an increase in Bacillota and Clostridia, differed from that of healthy horses without crib-biting, consistent with findings from previous studies. Furthermore, metagenome prediction suggests metabolic profile changes in bacterial communities between both conditions in horses. Further studies are required to validate the role of the microbiota-gut-brain axis in the etiology of crib-biting and other abnormal and stereotyped behaviors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Domestic horses could be bred for leisure activities and meat production, as is already the case in many countries. Horse meat is consumed in various countries, including Kazakhstan and Kyrgyzstan, and with the increase in this consumption, horses are registered as livestock by the Food and Agricultural Organization. In this study, horse meat microbiota of horse samples ( n = 56; 32 samples from Kazakhstan and 24 samples from Kyrgyzstan) from two countries, Kazakhstan ( n = 3) and Kyrgyzstan ( n = 1), were investigated for the first time by next-generation sequencing and metabarcoding analysis. The results demonstrated that Firmicutes, Proteobacteria, and Actinobacteria were the dominant bacterial phyla in all samples. In addition, three (5.4%) Staphylococcus strains were isolated from the Uzynagash region, Kazakhstan. Staphylococcus strains were identified as Staphylococcus warneri , S. epidermidis , and S. pasteuri by partial 16S rRNA DNA gene Sanger sequencing. All three Staphylococcus isolates were nonbiofilm formers; only the S. pasteuri was detected as multidrug-resistant (resistant to penicillin, cefoxitin, and oxacillin). In addition, S. pasteuri was found to carry mec A, mec C, and tet K genes. This is the first study to detect potentially pathogenic Staphylococcus spp. in horse meat samples originating from Kazakhstan. In conclusion, it should be carefully considered that undercooked horse meat may pose a risk to consumers in terms of pathogens such as antibiotic-resistant Staphylococcus isolates.

The presence of fungi in tracheal wash (TW) of horses was recently linked to mild-moderate equine asthma, indicating a possible causal role; however, increased numbers of fungi may also stem from asthma-related alteration of tracheal mucus clearance or from environmental exposure. Our objective was to elucidate the association between the presence of fungi in TW and asthma status while controlling for relevant confounders. We conducted a retrospective case-control study involving 73 horses, including 34 controls and 39 asthmatic cases. Each asthmatic horse was matched with a control from the same barn to account for the influence of environmental exposure. All horses underwent respiratory clinical scoring, endoscopy, TW, and bronchoalveolar lavage (BAL). The association between asthma status and presence of TW fungi was tested with multivariable logistic regression modelling, accounting for selected management factors, tracheal mucus accumulation, and selected TW and BAL cytological characteristics, including multinucleated giant cells (MGCs) in the TW. Given the variability in MGC definitions in the literature, particularly concerning their morphology and number of nuclei, we constructed two distinct models for each outcome (asthma status or presence of fungi in TW): one considering MGCs as cells with ≥ 3 nuclei, and another using a criterion of ≥ 10 nuclei. Horses with a tracheal mucus score ≥ 2 exhibited 3.6 to 4.3 higher odds of being asthmatic, depending on the MGC definition. None of the other variables examined were associated with either asthma status or TW fungi detection. Notably, the presence of fungal elements in the TW was not associated with equine asthma., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dély et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glanders, a highly contagious and often fatal disease affecting equids, is caused by Burkholderia mallei. Although sporadic cases of equine glanders have recently been documented in Mongolia, genome sequencing and molecular studies of the bacteria within this region are lacking. This study provided the first molecular characterization of B. mallei isolated from four native Mongolian horses from two different provinces in 2019 and 2022 by applying whole-genome sequencing with two SNP types (previously developed genotyping with 15 SNP markers that provide global coverage of the B. mallei population and the core genome coding SNP typing developed in this study). The Mongolian isolates were located within the L3B1 cluster, which was previously associated with the V-120 strain from Russia. Within the L3B1 cluster shared by neighboring countries, they were in a unique subbranch. In this study, specific SNP markers unique to the Mongolian strains were identified to track these strains using a high-resolution melting analysis (HRMA). This study revealed the unique phylogenetic background of Mongolian strains isolated from the eastern part of Mongolia. HRMA specific to the Mongolian subbranch may contribute to the molecular epidemiological monitoring of glanders in Mongolia and surrounding countries., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Understanding normal microbial populations within areas of the respiratory tract is essential, as variable regional conditions create different niches for microbial flora, and proliferation of commensal microbes likely contributes to clinical respiratory disease. The objective was to describe microbial population variability between respiratory tract locations in healthy horses. Samples were collected from four healthy adult horses by nasopharyngeal lavage (NPL), transtracheal aspirate (TTA), and bronchoalveolar lavage (BAL) of six distinct regions within the lung. Full-length 16S ribosomal DNA sequencing and microbial profiling analysis was performed. There was a large amount of diversity, with over 1797 ASVs identified, reduced to 94 taxa after tip agglomeration and prevalence filtering. Number of taxa and diversity were highly variable across horses, sample types, and BAL locations. Firmicutes, proteobacteria, and actinobacteria were the predominant phyla. There was a significant difference in richness (Chao1, p = 0.02) and phylogenetic diversity (FaithPD, p = 0.01) between NPL, TTA, and BAL. Sample type (p = 0.03) and horse (p = 0.005) contributed significantly to Bray-Curtis compositional diversity, while Weighted Unifrac metric was only affected by simplified sample type (NPL and TTA vs BAL, p = 0.04). There was no significant effect of BAL locations within the lung with alpha or beta diversity statistical tests. Overall findings support diverse microbial populations that were variable between upper and lower respiratory tract locations, but with no apparent difference in microbial populations of the six biogeographic regions of the lung, suggesting that BAL fluid obtained blindly by standard clinical techniques may be sufficient for future studies in healthy horses., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the gastrointestinal mucosa, there is a close cooperation between secretory immunoglobulin A (SIgA) and the composition of the microbiota, which aims to maintain homeostasis as well as act as a protective barrier. The purpose of this study was to determine the composition of microbiota and SIgA production in different parts of the digestive tract (small intestine, cecum, colon and rectum) of nine healthy horses and its reflection in the feces. For this purpose, we determined: the composition of the microbiome (by next-generation Sequencing of Hypervariable Regions V3-V4 and V7-V9 of the 16 S rRNA gene analysis), the amount of SIgA in the intestinal content samples (by ELISA), as well as the number of IgA-producing cells (IgA+) in the tissue samples (by immohistochemical analysis). Significant differences were observed between the small intestine and the large colon in the composition and diversity of the microbiome, as well as the number of IgA + cells in the mucosal lamina propria and the abundance of SIgA in the intestinal lumen. The small intestine in relation to the large colon is characterised by fewer IgA + cells, more SIgA in the intestinal contents and a less diverse microbiome. However, the cecum appears to be the third separate ecosystem, with a high number of IgA + cells and a diverse microbiome. The fecal sample reflects the current state of the large colon, both in terms of the microbiome and SIgA content; however, it is not known to what extent it may be influenced by dysbiosis in other parts of the digestive tract., (© 2024. The Author(s).)

Objective: To determine the ability of bacteria commonly isolated from equine limb wounds to survive in saltwater obtained from an equine hydrotherapy unit at different salinity concentrations and temperatures., Methods: Saltwater samples were obtained over a 2-week period (January 22, 2024 to February 2, 2024) from an equine hydrotherapy unit used for clinical patients, kept at either full salinity per manufacturer recommendations or diluted to half salinity to mimic the dilution that likely occurs in the clinical setting between cases when holding tanks are replenished with tap water only. Samples were then autoclaved to eliminate preexisting bacterial contamination before individual inoculation with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus zooepidemicus. Each inoculated sample was maintained at 2, 22, or 44 °C to represent typical holding tank conditions. The bacterial concentration was determined at each condition every 24 hours up to and including 96 hours. The lower limit of detection was set at 1 CFU/mL., Results: Salinity did not affect bacterial survival. Bacterial concentrations generally decreased with increasing temperature over time. Escherichia coli, S aureus, and S zooepidemicus concentrations decreased to the lower limit of detection at 44 °C by 24 to 48 hours, while P aeruginosa concentrations significantly decreased over 24 hours but remained well above the lower limit of detection., Conclusions: Common bacterial isolates of equine limb wounds can survive in typical saltwater hydrotherapy conditions., Clinical Relevance: Further investigation is warranted to determine the clinical relevance of these findings including protocols for hydrotherapy unit disinfection, wastage of treatment water, and case inclusion/exclusion criteria.

Background: The microbial composition of Sumbawa Horse Milk is influenced by various factors, including environmental elements that encompass geographical location, climate, and conditions specific to Sumbawa. This study aimed to determine the biodiversity and genetic diversity of the microbiome of Sumbawa Horse Milk, with an emphasis on yeast., Methods: The diversity and group of yeast isolates were evaluated by the sequence-related amplified polymorphism (SRAP) method using ME2F-EM15R (1) and ME2F-EM12R (2) primers. Molecular identification using 18 S rRNA primers was then carried out on nine selected isolates (K&#95;21, K&#95;31, K&#95;42, K&#95;45, K&#95;1, K&#95;6, K&#95;8, K&#95;17, and K&#95;19) to determine the type of yeast. Probiotic candidate tests were carried out on three isolates, namely K&#95;1, K&#95;6 and K&#95;8., Results: Analysis with NTSYS software on the SRAP results using Primer 1 revealed the presence of two major groups, where Group I was exclusively comprised of K&#95;45 isolate, whereas the other isolates belonged to Group II. On the other hand, analysis with NTSYS software on the SRAP analysis with Primer 2 also showed two major groups with different compositions. Group I consisted of isolates K&#95;39, 38, 37, 36, 35, 34, 33, 31, 29, 28, 27, 26, 25, 24, 23, 22, and 21, while the remaining isolates belonged to Group II. Results of 18 S rRNA analysis demonstrated that K&#95;17 and K&#95;19 had 99.8 and 100% similarity, respectively, and identified as Candida humilis. K&#95;21, K&#95;31, and K&#95;45 were identified as having a 100% similarity to Clavispora lusitaniae, while K&#95;42 had a 99.8% similarity to Candida parapsilosis. Three isolates were identified as belonging to the genus Ogataea, namely Ogataea polymorpha (K&#95;6 and K&#95;8) and Ogataea siamensis (K&#95;1) with similarity of 100% and 99.8%, respectively., Conclusions: These findings suggest that the three yeast have potential as probiotics., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by internal transcribed spacer sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silvicola (n = 1) and Moesziomyces aphidis (n = 1), respectively. The six H. capsulatum sensu lato isolates were further characterised by multilocus sequence analysis. Four distinct gene loci (arf [462 bases], H-anti [410 bases], ole1 [338 bases] and tub1 [272 bases]) of these six isolates as well as those of two H. capsulatum sensu lato ('var. farciminosum') reference strains (ATCC 58332 and ATCC 28798) were polymerase chain reaction (PCR)-amplified and sequenced. Phylogenetic analyses of their concatenated nucleotide sequences showed that three of the isolates and the reference strain ATCC 58332 were identical and belonged to the Eurasia clade within Latin American (LAm) A (H. suramericanum), and those of the other three isolates and the reference strain ATCC 28798 were identical and belonged to the Africa clade. At least two distinct phylogenetic clades of H. capsulatum sensu lato were circulating in Ethiopian horses with EL. Advanced molecular technologies and bioinformatics tools are crucial for the accurate identification and typing of pathogens as well as the discovery of novel microorganisms in veterinary microbiology., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Studies on the bacterial composition of seminal samples have primarily focused on species isolated from semen and their effects on fertility and reproductive health. Culture-independent techniques, such as 16S rRNA gene sequencing and shotgun metagenomics, have revolutionized our ability to identify unculturable bacteria, which comprise >90% of the microbiome. These techniques allow for comprehensive analysis of microbial communities in seminal samples, shedding light on their interactions and roles. In this study, we characterized the taxonomic diversity of seminal microbial communities in healthy stallions using 16S rRNA gene sequencing. Semen samples were collected from four stallions during the reproductive season, and DNA was extracted for sequencing. The results revealed a diverse array of bacterial taxa, with Firmicutes, Bacteroidota, and Proteobacteria being predominant phyla. At the family and genus levels, significant variations were observed among individuals, with individual variability in microbial richness and diversity standing out. Moreover, each stallion showed a distinct microbial fingerprint, indicating the presence of a characteristic microbial core for each stallion. These results underscore the importance of considering individual microbial profiles in understanding reproductive health and fertility outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Leptospirosis is an infectious disease with a worldwide distribution, which represents a major challenge in animal production across developing countries, mainly in tropical areas. Horses are particularly susceptible to the disease, presenting manifestations ranging from subclinical to the development of uveitis that compromises the visual health of the animals. In recent years, serological studies have been carried out in equid populations from America, demonstrating high exposure. For this reason, the aim of this study was to demonstrate microbiologically and molecularly the presence of the members of the genus Leptospira in urine samples from equids in an endemic state of leptospirosis in Mexico, and to detect the serological presence of anti-Leptospira antibodies in the sampled animals. For this reason, blood and urine samples were collected from 28 horses and one mule from three localities in the state of Veracruz, Mexico. Urine samples were inoculated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and the recovered isolates were typed using a short Multi Locus Sequence Typing scheme. Amplifications of the expected size were subjected to sequencing, and the recovered sequences were compared with those of reference deposited in GenBank using the BLAST tool. To identify their phylogenetic position, we performed a phylogenetic reconstruction using the maximum likelihood method. Additionally, Microscopic Agglutination test was performed on the serum samples to identify anti-Leptospira antibodies. We recovered 16 urine isolates which tested positive for the presence of Leptospira DNA. The phylogenetic reconstruction and the MLST analysis confirmed the presence of several genotypes of Leptospira interrogans and Leptospira santarosai. An overall serological frequency of 97.1 % was detected. Our results represent the first record of the presence of Leptospira through bacteriological isolates in equids from Mexico., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Characterization of microbiota structure on the skin of healthy horses is important for further development of modulation strategies to ensure optimal bacterial composition for physiological processes. This requirement is also supported by the relatively high incidence of dermatological diseases in horses and thus the need to manage them therapeutically. The taxonomic analysis of skin samples (n = 30) from five different body parts of clinically healthy Shetlands ponies females (neck, back, abdomen, pastern, muzzle) kept under homogeneous conditions (in open stalls with paddock, feed with dry hay, green grass ad libitum and granulated feed) was performed using amplification of V3-V4 region of the 16S rRNA gene. Results indicate that bacteria associated with healthy equine skin represent 18 phyla, 29 classes and 119 families. The most abundant phyla were Proteobacteria (30.8 ± 9.1%) followed by Actinobacteriota (20.4 ± 7.6%), Firmicutes (19.5 ± 10.1%), Bacteroidota (8.5 ± 5.0%) and Deinococcota (7.2 ± 14.8%). Among 229 genera identified, Corynebacterium (7.4 ± 6.5%) was the most abundant genus in skin sites of horses, followed by Deinococcus (7.1 ± 14.9%) and Macrococcus (5.0 ± 8.2%). Indices for the richness and diversity of species within bacterial populations for five regions of horses skin revealed no significant variations observed for species richness (Chao1, p-value 0.2001) but significant result for species evenness (Shannon, p-value 0.0049) with maximum on the neck and minimum on the back skin site. The clustering was seen across samples from different skin sites but also across samples collected from individual horses., (© 2024. The Author(s).)

Gut microbiomes are widely hypothesised to influence host fitness and have been experimentally shown to affect host health and phenotypes under laboratory conditions. However, the extent to which they do so in free-living animal populations and the proximate mechanisms involved remain open questions. In this study, using long-term, individual-based life history and shallow shotgun metagenomic sequencing data (2394 fecal samples from 794 individuals collected between 2013-2019), we quantify relationships between gut microbiome variation and survival in a feral population of horses under natural food limitation (Sable Island, Canada), and test metagenome-derived predictions using short-chain fatty acid data. We report detailed evidence that variation in the gut microbiome is associated with a host fitness proxy in nature and outline hypotheses of pathogenesis and methanogenesis as key causal mechanisms which may underlie such patterns in feral horses, and perhaps, wild herbivores more generally., (© 2024. The Author(s).)

Background: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region., Results: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities., Conclusions: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome., (© 2024. The Author(s).)

An international collection of Staphylococcus aureus of clonal complex (CC) 398 from diverse hosts spanning all continents and a 30 year-period is studied based on whole-genome sequencing (WGS) data. The collection consists of publicly available genomic data from 2994 strains and 134 recently sequenced Swiss methicillin-resistant S. aureus (MRSA) CC398 strains. A time-calibrated phylogeny reveals the presence of distinct phylogroups present in Asia, North and South America and Europe. European MRSA diverged from methicillin-susceptible S. aureus (MSSA) at the beginning of the 1950s. Two major European phylogroups (EP4 and EP5), which diverged approximately 1974, are the main drivers of MRSA CC398 spread in Europe. Within EP5, an emergent MRSA lineage spreading among the European horse population (EP5-Leq) diverged approximately 1996 from the pig lineage (EP5-Lpg), and also contains human-related strains. EP5-Leq is characterized by staphylococcal cassette chromosome mec (SCCmec) IVa and spa type t011 (CC398-IVa-t011), and EP5-Lpg by CC398-SCCmecVc-t011. The lineage-specific antibiotic resistance and virulence gene patterns are mostly mediated by the acquisition of mobile genetic elements like SCCmec, S. aureus Genomic Islands (SaGIs), prophages and transposons. Different combinations of virulence factors are present on S. aureus pathogenicity islands (SaPIs), and novel antimicrobial resistance gene containing elements are associated with certain lineages expanding in Europe. This WGS-based analysis reveals the actual evolutionary trajectory and epidemiological trend of the international MRSA CC398 population considering host, temporal, geographical and molecular factors. It provides a baseline for global WGS-based One-Health studies of adaptive evolution of MRSA CC398 as well as for local outbreak investigations., (© 2024. The Author(s).)

Horse owners and veterinarians report that from the age of 15, their horses can lose body condition and be more susceptible to diseases. Large intestinal microbiome changes may be involved. Indeed, microbiota is crucial for maintaining the condition and health of herbivores by converting fibres into nutrients. This study aimed to compare the faecal microbiome in horses aged from 6 to 30 years old (yo), living in the same environment and consuming the same diet, in order to assess whether the parameters changed linearly with age and whether there was a pivotal age category. Fifty horses were selected from the same environment and distributed across four age categories: 6-10 (n = 12), 11-15 (n = 11), 16-20 (n = 13), and 21-30 (n = 14) yo. All horses had no digestive problems, had teeth suitable for consuming their feed, and were up to date with their vaccination and deworming programmes. After three weeks of constant diet (ad libitum hay and 860 g of concentrate per day), one faecal sample per horse was collected on the same day. The bacterial communities' richness and intra-sample diversity were negatively correlated with age. There was a new distribution of non-beneficial and beneficial taxa, particularly in the 21-30 yo category. Although the faecal concentration of short-chain fatty acids remained stable, the acetate proportion was negatively correlated with age while it was the opposite for the proportions of butyrate, valerate, and iso-valerate. Additionally, the faecal pH was negatively correlated with age. Differences were more pronounced when comparing the 6-10 yo and 21-30 yo categories. The values of the parameters studied became more dispersed from the 16-20 yo category onwards, which appeared as a transitional moment, as it did not differ significantly from the younger and older categories for most of these parameters. Our data suggest that the microbiome changes with age. By highlighting the pivotal age of 16-20, this gives the opportunity to intervene before individuals reach extremes that could lead to pathological conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baraille et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Methicillin resistant Staphylococcus aureus (MRSA) represents a worrying example of antimicrobial resistance, and it is essential to acquire new information to monitor the spread and limit it further diffusion. This study aimed to characterise 22 MRSA isolates from horses, dogs, cats, and their human handlers focusing on spa typing. In the analysis of the sequences obtained, the spa type is "unknown" (unidentified) and all the sequences except one had repeats previously not known in all databases potentially indicating new spa-repeats. This could possibly indicate either permanent import of novel spa types or in-house microevolution of spa repeats., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The microbiome plays a significant role in shaping the health and functioning of the systems it inhabits. The seminal microbiome of stallions has implications for the health of the reproductive tract, sperm quality during preservation and antibiotic use in semen extenders. Diverse bacteria are present on the external genital tract and a mix of commensal microorganisms populates various parts of the reproductive tract, influencing the seminal bacterial content. Other sources of bacteria include the environment, semen collection equipment, and personnel. The bacterial load can adversely affect sperm quality and fertility, particularly in artificial insemination, where semen is extended and stored before use. Antibiotics are frequently used to inhibit bacterial growth, but their effectiveness varies depending on the bacterial strains present. The aim of this study was to assess the bacterial diversity in semen from 37 healthy stallions across three European nations (Germany, Portugal, and Sweden) using 16S sequencing. Semen samples were collected from individual stallions at three AI centers; DNA extraction, sequencing, and bioinformatic analysis were performed. Differences in bacterial diversity among the stallions were seen; although bacterial phyla were shared across the regions, differences were observed at the genus level. Climate, husbandry practices, and individual variability likely contribute to these differences. These findings underscore the importance of tailoring antibiotic strategies for semen preservation based on regional bacterial profiles. The study presents a comprehensive approach to understanding the intricacies of the stallion seminal microbiome and its potential implications for reproductive technologies and animal health., (© 2024. The Author(s).)

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a mucosal commensal of the lower genital tract in horses and is the most isolated bacterium causing endometritis in mares. The aim of this study was to determine the molecular diversity of S. zooepidemicus obtained from endometritis in mares in Buenos Aires province, Argentina. Thirty isolates obtained from the uterus of mares in 2005 and 2017 were studied. The MLST scheme was applied to identify the Argentinian genotypes and the clonal relationships and patterns of evolutionary descent were identified using the eBURST algorithm - goeBURST. Twenty six different Sequence types (STs) were identified, being only 11 of them previously reported in horses and also, from several host species and tissues. The other 15 STs were reported in Argentinian reproductive strains of mares in our study for the first time. The genotypes obtained from uterus in Argentina were not evenly distributed when all the published S. zooepidemicus STs were analysed, thus, it was not possible to establish that the same lineage circulates in our equine population. The fact that the identified genotypes were also reported in other countries, diverse samples and host species suggest that there is not a host, and an anatomical niche adaptation. Finally, the isolation of the same genotype in the vagina/clitoris and the uterus of the same mare highlights the versatility of S. zooepidemicus and its role as an opportunistic pathogen., Competing Interests: Declaration of competing interest None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence the content of this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020-2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To determine the effects of astaxanthin (ASTX) supplementation on the equine gut microbiota during a deconditioning-reconditioning cycle, 12 polo ponies were assigned to a control (CON; n = 6) or supplemented (ASTX; 75 mg ASTX daily orally; n = 6) group. All horses underwent a 16-week deconditioning period, with no forced exercise, followed by a 16-week reconditioning program where physical activity gradually increased. Fecal samples were obtained at the beginning of the study (Baseline), after deconditioning (PostDecon), after reconditioning (PostRecon), and 16 weeks after the cessation of ASTX supplementation (Washout). Following DNA extraction from fecal samples, v4 of 16S was amplified and sequenced to determine operational taxonomic unit tables and α-diversity and β-diversity indices. The total number of observed species was greater at Baseline than PostDecon, PostRecon, and Washout (p ≤ 0.02). A main effect of ASTX (p = 0.01) and timepoint (p = 0.01) was observed on β-diversity, yet the variability of timepoint was greater (13%) than ASTX (6%), indicating a greater effect of timepoint than ASTX. Deconditioning and reconditioning periods affected the abundance of the Bacteroidetes and Fibrobacteres phyla. Physical activity and ASTX supplementation affect the equine gut microbiome, yet conditioning status may have a greater impact., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The biopreservation strategy allows extending the shelf life and food safety through the use of indigenous or controlled microbiota and their antimicrobial compounds. The aim of this work was to characterize an inhibitory substance with bacteriocin-like activity (Sak-59) produced by the potentially probiotic L. sakei strain from artisanal traditional Kazakh horse meat product Kazy. The maximum production of Sak-59 occurred at the stationary phase of the L. sakei growth. Sak-59 showed inhibitory activity against gram-positive meat spoilage bacteria strains of Listeria monocytogenes, Staphylococcus aureus, and pathogenic gram-negative bacteria strains of Serratia marcescens and Escherichia coli, but not against the tested Lactobacilli strains. Sak-59 activity, as measured by diffusion assay in agar wells, was completely suppressed after treatment with proteolytic enzymes and remained stable after treatment with α-amylase and lipase, indicating that Sak-59 is a peptide and most likely not glycosylated or lipidated. It was concluded that Sak-59 is a potential new bacteriocin with a characteristic activity spectrum, which can be useful in the food and feed industries., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

A Gram-stain-negative, aerobic, non-endospore-forming organism isolated from horse blood was studied for its taxonomic allocation. On the basis of 16S rRNA gene sequence similarity comparisons, strain M6-77 T grouped within the genus Devosia and was most closely related to Devosia elaeis (97.6 %) and Devosia indica (97.55 %). The 16S rRNA gene sequence similarity to type strains of other Devosia species was below 97.5 %. The average nucleotide identity and digital DNA-DNA hybridization values between the M6-77 T genome assembly and those of the closest relative Devosia type strains were <85 and <25 %, respectively. Strain M6-77 T grew optimally at 25-37 °C (range: 10-36 °C), at a pH range of pH 6.5-10.5 and in the presence of up to 3 % (w/v) NaCl. The fatty acid profile from whole-cell hydrolysates supported the allocation of the strain to the genus Devosia . Major fatty acids were C 18 : 1  ω 7 c, 11-methyl C 18 : 1  ω 7 c and C 16 : 0 . The quinone system consisted exclusively of ubiquinone Q-10. The polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol and three unidentified glycolipids. In the polyamine pattern, putrescine was predominant and spermidine was detected in moderate amounts. The diamino acid of the peptidoglycan was meso- diaminopimelic acid. In addition, the results of physiological and biochemical tests also allowed phenotypic differentiation of strain M6-77 T from the closely related species. Hence, M6-77 T represents a new species of the genus Devosia , for which we propose the name Devosia equisanguinis sp. nov., with M6-77 T (=CIP 111628 T =LMG 30659 T =CCM 8868 T ) as the type strain.

Background: There is little objective information concerning the effect of steam-flaked grains on foal's growth performance and faecal microbiota. To determine the effects of steam-flaked grains on foal's growth performance and faecal microbiota, faecal samples were collection from 18 foals which had been fed either corn, oat or barley diets over the 60 days of the experiment. Body weight and conformation measurements were collected. Next-generation sequencing of the V3 + V4 region of the 16 S rRNA gene was used to assess the microbial composition of faeces. Alpha diversity, Venn graph, Relative abundance and beta diversity are presented., Results: There was a significantly higher larger increase in the body weight of those foals fed barley compared to either corn or oats. There were also significant changes in the Alpha diversity of the gut microbiota. The Shannon and Simpson indices were significantly higher in the barley fed group than those fed corn or oats. The Chao1 index was significantly higher in the oat fed group than the corn or barley fed groups. There were significant changes in the relative abundance of bacteria in the microbiota in terms of phylum, family and genus. The histogram of LDA value distribution showed that the 12 statistically different biomarkers of the bacteria were present. Tax4Fun function annotation clustering heat map showed that functional information was detected from 26 species of bacteria in faecal samples from the foals., Conclusions: Differences by starch sources were found in overall growth of the foals and in the faecal microbiota if either supplementary corn, oat or barley was fed. Further studies are required to determine the potential impact of the changes in the microbiota on the health and development of foals fed cereal starch of different sources., (© 2021. The Author(s).)

Antibiotic resistance in bacterial pathogens is a growing problem for both human and veterinary medicine. Mobile genetic elements (MGEs) such as plasmids, transposons, and integrons enable the spread of antibiotic resistance genes (ARGs) among bacteria, and the overuse of antibiotics drives this process by providing the selection pressure for resistance genes to establish and persist in bacterial populations. Because bacteria, MGEs, and resistance genes can readily spread between different ecological compartments (e.g. soil, plants, animals, humans, wastewater), a "One Health" approach is needed to combat this problem. The equine hindgut is an understudied but potentially significant reservoir of ARGs and MGEs, since horses have close contact with humans, their manure is used in agriculture, they have a dense microbiome of both bacteria and fungi, and many antimicrobials used for equine treatment are also used in human medicine. Here, we collate information to date about resistance genes, plasmids, and class 1 integrons from equine-derived bacteria, we discuss why the equine hindgut deserves increased attention as a potential reservoir of ARGs, and we suggest ways to minimize the selection for ARGs in horses, in order to prevent their spread to the wider community.

Infectious keratoconjunctivitis (IKC) is the most frequent ocular disease in livestock worldwide and is primarily caused by Moraxella bovis, M. ovis, and/or M. bovoculi. The economic impact of IKC is mainly due to ocular damage, which leads to weight loss, management difficulties, pain and discomfort, and cost of treatments. In horses, limited information is available on the association of Moraxella spp. with keratoconjunctivitis. The present report describes two cases of equine keratoconjunctivitis caused by members of the genus Moraxella. Both animals presented with lacrimation, conjunctivitis, photophobia, mucoid or purulent secretions, blepharitis, and conjunctival hyperemia. The diagnosis of IKC was based on the epidemiological and clinical findings; the etiological agent was identified through bacteriological (culture and biochemistry assays) and molecular testing (PCR and nucleotide sequencing). Our study reports the isolation of Moraxella bovoculi (SBP 88/19) and a putative new species/mutant of Moraxella (SBP 39/19) recovered from ocular secretions in horses. Thus, we suggest the inclusion of Moraxella spp. infection in the differential diagnosis of conjunctivitis in horses in Southern Brazil., (© 2021. Sociedade Brasileira de Microbiologia.)

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide β-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Gerald B. Pier (GBP) is an inventor of intellectual properties [human monoclonal antibody to PNAG and PNAG vaccines] that are licensed by Brigham and Women’s Hospital to Alopexx Vaccine, LLC, an entity in which GBP also holds equity. As an inventor of intellectual properties, GBP also has the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women’s Hospital from Alopexx Vaccine, LLC. GBP’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners Health care in accordance with their conflict of interest policies. Colette Cywes-Bentley (CC-B) is an inventor of intellectual properties [use of human monoclonal antibody to PNAG and use of PNAG vaccines] that are licensed by Brigham and Women’s Hospital to Alopexx Vaccine, LLC. As an inventor of intellectual properties, CC-B also has the right to receive a share of licensing-related income (royalties, fees) through Brigham and Women’s Hospital from Alopexx Vaccine, LLC. Daniel R. Vlock is the Chief Executive Officer of ALOPEXX Vaccines and owns the rights to the PNAG vaccine. Dr. Vlock had no role in the design or analysis of the study. SC Meyer and PJ Sutter work for MG Biologics that produced the plasma and thus might have potential earnings; however, they had no part in the design or analysis of the study. Drs. Glenn Blodgett and Nathan Canaday are employees of the 6666 Ranch and Dr. Carly Turner-Garcia is employed by the Lazy E Ranch. Sarah Meyer and Patrick Sutter are employed by Mg Biologics, Inc. and Dr. Daniel Vlock is employed by ALOPEXX Vaccines. The 6666 Ranch, Lazy E Ranch, Mg Biologics, and ALOPEXX provided support in the form of salaries, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ticks account for an extensive range of health and welfare issues in horses. In addition, tick-borne pathogens (TBPs) limit global animal trading and equine sporting events. Here, we assess the prevalence, co-infectivity and risk factors of TBPs in horse ticks in Korea. A total of 245 hard ticks, including 103 male and 142 female adults, were obtained from horses on Jeju Island during the spring to autumn seasons of 2013-2019. All collected ticks were identified as adult Haemaphysalis longicornis. We screened and analyzed each tick for the presence of several TBPs by polymerase chain reaction (PCR) analysis. Among the 245 ticks, we detected genes for three TBPs, Candidatus Rickettsia longicornii (22.9%), Ehrlichia canis (0.4%) and Theileria luwenshuni (0.4%), while Anaplasma spp. was not detected. TBPs were most prevalent in ticks harvested during the autumn season, and more abundant in the female than male adults. This is the first report of the genera Ehrlichia, Rickettsia and Theileria in horse ticks in Korea. TBPs in horse ticks are likely a reservoir for zoonotic transmission to other animals, including humans. Our findings demonstrate the need for further understanding of the prevalence and epidemiology of TBPs in wild and domestic animals., (© 2020 The Royal Entomological Society.)

Spotted fever group (SFG) rickettsiae are obligatory intracellular bacteria that cause disease in humans and other animals. Ixodid ticks are the principal vectors of SFG rickettsiae. The present study aimed to determine the prevalence and species identity of SFG rickettsiae in ticks and horses from urban and rural areas of western Cuba using PCR assays. Tick samples, collected from 79 horses, consisted of 14 Amblyomma mixtum adults, 111 Dermacentor nitens adults and 19 pools of D. nitens nymphs (2-5 individuals/pool). The PCR results revealed the presence of Rickettsia spp. in 64% of the A. mixtum adults, 16% of the D. nitens adults, and 11% of the pooled samples of D. nitens nymphs. In contrast, Rickettsia spp. was not detected in any of the 200 horse blood samples included in this study. DNA sequence data of the rickettsial 17 kDa antigen gene showed that Rickettsia amblyommatis was present in A. mixtum; and Rickettsia felis in D. nitens. This is the first report of R. felis in D. nitens in Cuba. The present study extends our knowledge of the potential vector spectrum and distribution of SFG rickettsiae pathogens in western Cuba., (© 2020 The Royal Entomological Society.)

Horse botflies have been a threat to the Przewalski's horses in the Kalamaili Nature Reserve in Xinjiang of China since their reintroduction to the original range. As larvae of these parasites could infest the intestine of a horse for months, they could interact with and alter the structure and composition of its intestinal microbiota, affecting adversely its health. Nonetheless, there are no such studies on the rewilded Przewalski's horses yet. For the first time, this study characterizes the composition of the intestinal microbiota of 7 rewilded Przewalski's horses infected severely by Gasterophilus pecorum following and prior to their anthelmintic treatment. Bioinformatics analyses of the sequence data obtained by amplicon high throughput sequencing of bacterial 16S rRNA genes showed that G. pecorum infestation significantly increased the richness of the intestinal microbial community but not its diversity. Firmicutes and Bacteroidetes were found the dominant phyla as in other animals, and the parasitic infestation decreased the F/B ratio largely by over 50%. Large reduction in relative abundances of the two genera Streptococcus and Lactobacillus observed with G. pecorum infestation suggested possible changes in colic and digestion related conditions of the infected horses. Variations on the relative abundance of the genus groups known to be pathogenic or symbiotic showed that adverse impact of the G. pecorum infestation could be associated with reduction of the symbiotic genera Lactobacillus and Bifidobacterium that are probiotics and able to promote immunity against parasitic infection., Competing Interests: The authors have declared that no competing interests exist.

Brazilian spotted fever (BSF) is a common tick-borne disease caused by Rickettsia rickettsii. Horses are the primary hosts of the main vector, Amblyomma sculptum, and are considered efficient sentinels for circulation of Rickettsia. Therefore, the aim of this study was to detect antibodies reactive to R. rickettsii antigens in horses from a non-endemic area in the north-central region of Bahia state, Brazil. Blood samples and ticks were collected from 70 horses from the municipalities of Senhor do Bonfim, Antônio Gonçalves, Pindobaçu, and Campo Formoso. The sera obtained were tested by an indirect immunofluorescence assay to detect anti-Rickettsia antibodies. Overall, 5.7% (4/70) of the horses reacted to R. rickettsii antigens. Ticks were collected from 18.6% (13/70) of the horses and were identified as A. sculptum and Dermacentor nitens. Despite being a non-endemic area, seropositive animals were identified in our study, suggesting circulation of rickettsial agents in the region. This is the first serological survey of this agent in horses from the north-central region of Bahia, and further studies are needed to understand the epidemiology of BSF in these locations.

Background: Transmission of antimicrobial resistant and virulent Escherichia coli (E. coli) from animal to human has been considered as a public health concern. This study aimed to determine the phylogenetic background and prevalence of diarrheagenic E. coli and antimicrobial resistance in healthy riding-horses in Iran. In this research, the genes related to six main pathotypes of E. coli were screened. Also, genotypic and phenotypic antimicrobial resistance against commonly used antibiotics were studied, then phylo-grouping was performed on all the isolates., Results: Out of 65 analyzed isolates, 29.23 % (n = 19) were determined as STEC and 6.15 % (n = 4) as potential EPEC. The most prevalent antimicrobial resistance phenotypes were against amoxicillin/clavulanic acid (46.2 %) and ceftriaxone (38.5 %). bla TEM was the most detected resistance gene (98.4 %) among the isolates and 26.15 % of the E. coli isolates were determined as multi-drug resistant (MDR). Three phylo-types including B1 (76.92 %), A (13.85 %) and D (3.08 %) were detected among the isolates., Conclusions: Due to the close interaction of horses and humans, these findings would place emphasis on the pathogenic and zoonotic potential of the equine strains and may help to design antimicrobial resistance stewardship programs to control the dissemination of virulent and multi-drug resistant E. coli strains in the community.

Elite horse athletes that live in individual boxes and train and compete for hours experience long-term physical and mental stress that compromises animal welfare and alters the gut microbiota. We therefore assessed if a temporary period out to pasture with conspecifics could improve animal welfare and in turn, favorably affect intestinal microbiota composition. A total of 27 athletes were monitored before and after a period of 1.5 months out to pasture, and their fecal microbiota and behavior profiles were compared to those of 18 horses kept in individual boxes. The overall diversity and microbiota composition of pasture and control individuals were temporally similar, suggesting resilience to environmental challenges. However, pasture exposure induced an increase in Ruminococcus and Coprococcus that lasted 1-month after the return to individual boxes, which may have promoted beneficial effects on health and welfare. Associations between the gut microbiota composition and behavior indicating poor welfare were established. Furthermore, withdrawn behavior was associated with the relative abundances of Lachnospiraceae AC2044 group and Clostridiales family XIII. Both accommodate a large part of butyrate-producing bacterial genera. While we cannot infer causality within this study, arguably, these findings suggest that management practices maintained over a longer period of time may moderate the behavior link to the gut ecosystem beyond its resilience potential.

In the present research work, we propose a new antimicrobial treatment for pyoderma via cutaneous permeation of bacteriophage particles conveyed in a hydroxyethylcellulose (HEC) gel integrating ionic liquid as a permeation enhancer. Ionic liquids are highly viscous fluids constituted exclusively by ions, that are usually hydrolytically stable and promote solubilization of amphipathic molecules such as proteins, hence serving as green solvents and promoting the transdermal permeation of biomolecules. In the research effort entertained herein, the synthesis and use of choline geranate for integrating a HEC gel aiming at the structural and functional stabilization of a cocktail of isolated lytic bacteriophage particles was sought, aiming at transdermal permeation in the antimicrobial treatment of animal pyoderma. The results obtained showed a high ability of the ionic liquid in enhancing transdermal permeation of the bacteriophage particles, with concomitant high potential of the HEC gel formulation in the antimicrobial treatment of animal skin infections., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Livestock play an important role as reservoir of enteric pathogens and antimicrobial resistance (AMR), a health and economic concern worldwide. However, little is known regarding the transmission and maintenance of these pathogens at the wildlife-livestock interface. In this study, we assessed the occurrence, genetic diversity and AMR of Campylobacter spp. and Salmonella spp. shed by sympatric free-ranging livestock and a wild herbivore in an alpine ecosystem., Results: Campylobacter spp. was isolated from 23.3 % of cattle and 7.7 % of sheep but was not isolated from horses nor Pyrenean chamois (Rupicapra pyrenaica). Campylobacter jejuni was the most frequent species. A high genetic diversity and certain host specificity of C. jejuni isolates was observed. The main AMR detected in Campylobacter isolates was to nalidixic acid (88.2 %), ciprofloxacin (82.4 %) and tetracycline (82.4 %); only 11.7 % of the isolates were pan-susceptible and 17.6 % were multi-resistant. Salmonella ser. Newport was isolated only from one Pyrenean chamois and was pan-susceptible., Conclusions: Results show that free-ranging cattle and sheep are spreaders of Campylobacter as well as their AMR strains in the alpine environment. Therefore, contaminated alpine pastures or streams may constitute a source for the dissemination of AMR enteropathogens. However, apparently, alpine wild ungulates such as Pyrenean chamois play a negligible role in the epidemiology of zoonotic enteropathogens and AMR, and are not potential bioindicators of the burden of alpine environments.

The eye is host to myriad bacterial, fungal, and viral organisms that likely influence ocular surface physiology in normal and diseased states. The ocular surface mycobiota of horses has not yet been described using NGS techniques. This study aimed to characterize the ocular surface fungal microbiota (mycobiota) in healthy horses in 2 environmental conditions (stalled versus pasture). Conjunctival swabs of both eyes were obtained from 7 adult stallions stabled in an open-air pavilion and 5 adult mares living on pasture. Genomic DNA was extracted from ocular surface swabs and sequenced using primers that target the Internal Transcribed Spacer 1 (ITS1) region of the fungal genome on an Illumina platform. Sequences were processed using Quantitative Insights Into Molecular Ecology (QIIME 2.0) and taxonomy assigned with the Findley et al. 2013 ITS1 database. The most abundant genera identified were Leptosphaerulina (22.7%), unclassified Pleosporaceae (17.3%), Cladosporium (16.2%), Alternaria (9.8%), unclassified Pleosporales (4.4%), unclassified Montagnulaceae (2.9%), Fusarium (2.5%), and Pestalotiopsis (1.4%). Fungal community composition (Jaccard, R = 0.460, p = 0.001) and structure (Bray-Curtis, R = 0.811, p = 0.001) were significantly different between pastured mares and stabled stallions. The ocular surface of pastured mares had significantly increased fungal species richness and diversity compared to stabled stallions (Shannon p = 0.0224, Chao1 p = 0.0118, Observed OTUs p = 0.0241). Relative abundances of Aspergillus (p = 0.005) and Alternaria spp. (p = 0.002) were significantly increased in the mycobiota of pastured mares. This is the first report to describe the mycobiota of the equine ocular surface. Environmental factors such as housing influence the composition, structure, and richness of the equine ocular surface mycobiota., Competing Interests: The authors have declared that no competing interests exist.

Pneumonia caused by the intracellular bacterium Rhodococcus equi is an important cause of disease and death in immunocompromised hosts, especially foals. Antibiotics are the standard of care for treating R. equi pneumonia in foals, and adjunctive therapies are needed. We tested whether nebulization with TLR agonists (PUL-042) in foals would improve innate immunity and reduce the severity and duration of pneumonia following R. equi infection. Neonatal foals (n = 48) were nebulized with either PUL-042 or vehicle, and their lung cells infected ex vivo. PUL-042 increased inflammatory cytokines in BAL fluid and alveolar macrophages after ex vivo infection with R. equi. Then, the in vivo effects of PUL-042 on clinical signs of pneumonia were examined in 22 additional foals after intrabronchial challenge with R. equi. Foals infected and nebulized with PUL-042 or vehicle alone had a shorter duration of clinical signs of pneumonia and smaller pulmonary lesions when compared to non-nebulized foals. Our results demonstrate that host-directed therapy can enhance neonatal immune responses against respiratory pathogens and reduce the duration and severity of R. equi pneumonia.

Prudent use of antimicrobials is paramount to slow the development of resistance and for successful treatment. The use of cumulative antibiograms will allow evidence-based antimicrobial selection with consideration of local resistance patterns. We generated a "first-isolate-per-patient" cumulative antibiogram for a regional equine referral hospital. Bacterial organisms cultured from horses between 2011 and 2018, sample origin, antimicrobial susceptibilities, and multidrug-resistant (MDR) status were tabulated. Of 1,176 samples, 50% were culture positive. Overall, 93 of 374 (25%) were MDR. Of the MDR isolates, 11 (12%) were susceptible to high-importance antimicrobials only (as defined by the Australian Strategic and Technical Advisory Group on antimicrobial resistance). β-hemolytic streptococci were uniformly susceptible to penicillin (76 of 76); 17 of 20 (85%) non-β-hemolytic Streptococcus spp. were susceptible to penicillin. Despite veterinary-specific challenges in constructing an antibiogram, our study provides an exemplar of the clinical utility of regional-, farm-, or hospital-specific cumulative antibiograms for evidence-based empirical antimicrobial selection by veterinarians prior to susceptibility result availability.

Rickettsia amblyommatis is widely distributed in the Americas, and has been reported to infect different species of ticks within its distribution. In Panama, R. amblyommatis is the most common Rickettsia and its presence was molecularly detected in nine species of ticks and one flea species. This work described the isolation of R. amblyommatis in Vero cells by shell vial technique, from Amblyomma mixtum ticks collected from a captive tapir from Gamboa (Colon province), and a horse from El Valle de Antón (Cocle province). These represent the first isolations of R. amblyommatis in Panama., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

We investigated the specificity and sensitivity of two horse-associated markers, HoF597 and Horse mtCytb, and 12 mitochondrial and bacterial markers of six animal species (human, cow, pig, bird, dog, chicken) in the faecal samples of 50 individual horses. Both horse markers were detected in 48 (96%) faecal samples. Cross-reactivity with dog (BacCan545) and pig (P23-2) occurred in 88% and 72% of horse faecal samples, respectively. Several other bacterial and mitochondrial markers of non-target hosts were also detected; however, their specificities were >80%. Analyses of samples from surface waters ( n = 11) on or adjacent to properties from which horse faecal samples had been collected showed only the presence of HoF597 but not horse mitochondrial marker. Our data suggest that while bacterial and (or) mitochondrial markers of other animal species may be present in horse faeces, dog and pig markers may predominantly be present in horse faecal samples, which points to their nonspecificity as markers for microbial source tracking. Although HoF597 and Horse mtCytb are highly sensitive and specific for the detection of horse faecal pollution, because of their low numbers, mitochondrial (mtDNA) markers may not be robust for screening surface waters.

We isolated and characterized 65 anaerobic gut fungal (AGF; Neocallimastigomycota) strains from fecal samples of five wild (W, axis deer, white-tailed deer, Boer goat, mouflon, and Nilgiri tahr), one zoo-housed (Z, zebra), and three domesticated (D,  horse, sheep, and goat) herbivores in the US states of Texas (TX) and Oklahoma (OK), Wales (WA), and the Indian states of Kerala (KE) and Haryana (HA). Phylogenetic assessment using the D1-D2 regions of the large subunit (28S) rDNA and internal transcribed spacer 1 (ITS1) identified seven monophyletic clades that are distinct from all currently recognized AGF genera. All strains displayed monocentric thalli and produced exclusively or predominantly monoflagellate zoospores, with the exception of axis deer strains, which produced polyflagellate zoospores. Analysis of amplicon-based AGF diversity surveys indicated that zebra and horse strains are representatives of uncultured AL1 group, whereas domesticated goat and sheep strains are representatives of uncultured AL5 group, previously encountered in fecal and rumen samples of multiple herbivores. The other five lineages, all of which were isolated from wild herbivores, have not been previously encountered in such surveys. Our results significantly expand the genus-level diversity within the Neocallimastigomycota and strongly suggest that wild herbivores represent a yet-untapped reservoir of AGF diversity. We propose seven novel genera and eight novel Neocallimastigomycota species to comprise these strains, for which we propose the names Agriosomyces longus (mouflon and wild Boer goat), Aklioshbomyces papillarum (white-tailed deer), Capellomyces foraminis (wild Boar goat), and C. elongatus (domesticated goat), Ghazallomyces constrictus (axis deer), Joblinomyces apicalis (domesticated goat and sheep), Khoyollomyces ramosus (zebra-horse), and Tahromyces munnarensis (Nilgiri tahr).

Bacteria colonize stallion semen during collection and processing which may cause disease in inseminated females or negatively affect sperm quality during storage prior to insemination. Antibiotics are added to semen extenders to control the growth of these bacteria but may induce antimicrobial resistance. Research into alternatives to antibiotics for this purpose requires knowledge of which bacteria are present in semen. Not all bacteria in semen, however, can be identified by conventional microbiological techniques. The objectives of the study were to: i) determine which bacteria are present in stallion semen using metagenomics; and ii) investigate individual differences in bacterial content in semen from all stallions on one premises. Bacterial DNA was extracted from ejaculates from seven stallions (one ejaculate per stallion) and bacteria were identified using 16S sequencing. In total, 83 bacterial genera were identified, varying from 25 to 52 among different individuals. There was a negative correlation (r = -0.81212; P <  0.05) between the presence of Treponema spp. and Advenella spp. In conclusion, most of the bacteria present in stallion semen could be identified to genus level by 16S sequencing even when present at a low frequency. This method of identification may help to clarify individual variation in bacterial content and its potential effects on fertility., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Abrupt dietary changes, as can be common when managing horses, may lead to compositional changes in gut microbiota, which may result in digestive or metabolic disturbances. The aim of this study was to describe and compare the faecal microbiota of ponies abruptly changed from pasture grazing ad libitum to a restricted hay-only diet and vice versa. The experiment consisted of two, 14-day periods. Faecal samples were collected on day 0 and days 1-3,7,14 after abrupt dietary change from grass to hay and from hay to grass. Microbial populations were characterised by sequencing the V3-V4 region of the 16S rRNA gene using the Illumina MiSeq platform, 4,777,315 sequences were obtained from 6 ponies. Further analyses were performed to characterise the microbiome as well as the relative abundance of microbiota present. The results of this study suggest that the faecal microbiota of mature ponies is highly diverse, and the relative abundances of individual taxa change in response to abrupt changes in diet. The faecal microbiota of ponies maintained on a restricted amount of hay-only was similar to that of the ponies fed solely grass ad libitum in terms of richness and phylogenetic diversity; however, it differed significantly in terms of the relative abundances at distinct taxonomic levels. Class Bacilli, order Lactobacillales, family Lactobacillaceae, and genus Lactobacillus were presented in increased relative abundance on day 2 after an abrupt dietary change from hay to grass compared to all other experimental days (P <0.05). Abrupt changes from grass to hay and vice versa affect the faecal microbial community structure; moreover, the order of dietary change appears to have a profound effect in the first few days following the transition. An abrupt dietary change from hay to grass may represent a higher risk for gut disturbances compared to abrupt change from grass to hay., Competing Interests: The authors have declared that no competing interests exist.

Background: Equine pythiosis is an emerging, devastating disease that is hard to treat. The tumour-like nodular skin masses grow rapidly and the outcome is generally fatal, and thus early diagnosis and intervention are important., Objectives: (i) To highlight the clinical, histological and haematological findings in pythiosis, and (ii) to evaluate the efficacy of direct sample multiplex-PCR targeting the single nucleotide polymorphisms within the ribosomal DNA region for detection and genotyping of Pythium insidiosum., Animals: Two hundred and twenty horses including 204 Arabian and 16 draft horses were surveyed., Methods: Case series study diagnosis was based on clinical, pathological and haematological findings typical of P. insidiosum infection, culture identification, immunohistochemical investigation and direct sample PCR., Results: The affected horses (24 of 220, 10.91%) presented with unifocal or multiple lesions on the abdomen, limbs, chest, face and mammary gland. Cases commonly had a history of access to stagnant water, ponds and intentionally flooded rice fields. Most were pregnant mares (58.33%). Histopathology revealed granulomatous reaction, blood vessel endotheliosis, heavy infiltration of eosinophils in the dermal layer, multifocal necrosis and Splendore-Hoeppli phenomenon. Unlike direct microscopy (50%) and culture (91.6%), multiplex-PCR assay identified P. insidiosum (Clade II) in all tested samples. To the best of the authors knowledge, this is the first study determining a clade of P. insidiosum causing equine pythiosis in Egypt., Conclusions and Clinical Importance: Direct sample multiplex-PCR assay is a potential tool for the early and rapid diagnosis of equine pythiosis. It overcomes limitations associated with morphological identification and provides a definitive diagnosis., (© 2020 ESVD and ACVD.)

The effect of dressings saturated with either a standardized suspension of probiotic bacteria or saline on healing of traumatic distal limb wounds in horses was evaluated for 24 days, and the systemic inflammatory effect was assessed. The wounds were divided in two groups based on the phase of healing: wounds with an incomplete (ICGB) or a complete granulation bed (CGB). The wound area was expressed as percentage of the wound area at day 0 and defined as relative wound area. The mean relative wound area decreased faster in probiotic than saline treated wounds. The difference was most obvious in CGB and increased rapidly from day 0 until day 12 up to 30%, and stabilized around 25% thereafter until the end of the observation period, but it was not statistically significant because of the large variation within the treatment groups. The mean wound area of CGB decreased to 28.4% (range: 6.3 to 49.3) with probiotic and to 51.9% (range: 29.3 to 81.7) with saline treatment at day 24. Additionally, the rate to 50% healing in CGB was 3.4 faster with probiotic compared to saline treatment, whereas in ICGB this was 1.9 faster. Topical probiotics did not increase serum amyloid A and white blood cell counts. Although the mentioned differences were not statistically significant, the clinical relevance of the effect of treatment with probiotics in CGB wounds is clear, supported by the differences in mean wound area in course of time and the time required to reach 50% healing (day 12 for probiotic vs more than day 24 for saline treated wounds). Thus the probiotic treated wounds reached 50% reduction in wound area in half of the time of the saline treated wounds. The topical use of probiotics can be considered as safe as it did not cause a systemic effect., Competing Interests: The study was funded by Micuri Aps, Denmark. Unfortunately the company does not exist anymore. Micuri has provided the materials required for the study, and payed for the bacteriology and the hematology. Micuri has payed a compensation to the contributing practitioners for their clinical work to treat the wounds of the horses. JW SL AJ have recieved an award for setting up the study and their practical work to perform the study (order of 50%). Analysis of the results and preparation of the manuscript has not been funded. Micuri Aps had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that there is no competing interests with Micuri Aps related to employment, consultancy, relationships, patents, products in development, marketed products etc. There is no relation to the PLOS ONE journal, no relation to any legal proceedings, and there is no benefit from publication of the paper. The funding by Micuri Aps does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data and/or materials.

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that causes caseous lymphadenitis, a disease that predominantly affects sheep, goat, cattle, buffalo, and horses, but has also been recognized in other animals. This bacterium generates a severe economic impact on countries producing meat. Gene expression studies using RNA-Seq are one of the most commonly used techniques to perform transcriptional experiments. Computational analysis of such data through reverse-engineering algorithms leads to a better understanding of the genome-wide complexity of gene interactomes, enabling the identification of genes having the most significant functions inferred by the activated stress response pathways. In this study, we identified the influential or causal genes from four RNA-Seq datasets from different stress conditions (high iron, low iron, acid, osmosis, and PH) in C. pseudotuberculosis , using a consensus-based network inference algorithm called miRsigand next identified the causal genes in the network using the miRinfluence tool, which is based on the influence diffusion model. We found that over 50% of the genes identified as influential had some essential cellular functions in the genomes. In the strains analyzed, most of the causal genes had crucial roles or participated in processes associated with the response to extracellular stresses, pathogenicity, membrane components, and essential genes. This research brings new insight into the understanding of virulence and infection by C. pseudotuberculosis .