Human tick-borne relapsing fever caused by Borrelia persica is common in western Asia. A survey of 301 horses in Israel revealed 9.96% seropositivity toward B. persica antigens; 1 horse (0.33%) was also PCR positive for B. persica DNA. Phylogenetic analysis supported a transmission cycle involving ticks, humans, and horses.

Amid the rising prevalence of antimicrobial resistance (AMR), attention is increasingly directed toward investigating additional factors that may contribute to or mediate its development, among which companion animals have emerged as a potential reservoir and transmission interface. Despite this, the contribution of free-roaming animal populations living in proximity to humans and domestic animals has yet to be thoroughly investigated worldwide. Accordingly, this study aimed to determine the prevalence and antimicrobial resistance patterns of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales in free-roaming cats in Israel. Rectal swabs were collected from 214 free-roaming cats admitted to two veterinary clinics between 2023 and 2024. ESBL-PE were recovered from 51 cats (23.8%), yielding 61 isolates, which were tested for susceptibility to multiple antimicrobials. Eleven bacterial species were identified, with Escherichia coli being the most frequently recovered (62.3%, 38/61). Nine purposively selected E. coli isolates underwent whole-genome sequencing (WGS). Multidrug resistance (MDR), defined as non-susceptibility to at least one agent in three or more antimicrobial categories, was observed in 72.1% (44/61) of isolates, and four isolates were carbapenemase-producing Enterobacterales (CPE). These findings suggest that free-roaming cats may serve as a previously overlooked ‘One Health’ link, possibly connecting environmental contamination to human and animal health risks.

Abstract Background Infections with extended‐spectrum beta‐lactamase‐producing Enterobacterales (ESBL‐PE) contribute to morbidity and mortality in human neonates. In foals, data are scarce. Objectives Determine the association between ESBL‐PE gastrointestinal colonization on hospital admission and infections in hospitalized neonates. Animals Sixty‐seven foals. Methods Prospective study of foals admitted to a veterinary hospital. Foals were screened for ESBL‐PE colonization and for infections. Risk factors and clinical outcomes were analyzed. Results Seventy‐six percent of foals suffered from at least 1 bacterial infection (n = 51/67). Sixty‐three non‐ESBL bacterial species and 19 ESBL‐PE species were isolated. Twenty foals (29.85%) had at least 1 hospital‐acquired infection (HAI) and 30 foals (44.8%) suffered from multidrug resistant infections. The prevalence rates of ESBL‐PE gastrointestinal colonization on admission and clinical ESBL HAIs were 47.8% (n = 32/67, 41 isolates) and 19.40% (n = 13/67, 19 isolates), respectively. On multivariable analysis, ESBL‐PE HAIs were associated with colonization on admission (P = .03, odds ratio [OR] = 4.60). In an outcome analysis, ESBL‐PE infection and HAIs were associated with surgery (ESBL‐PE infection: P = .04, OR = 4.70; HAI: P = .004, OR = 6.4) and HAI also was associated with increased duration of hospitalization (P < .001, OR = 9.13). The major colonizing and infecting bacterial species were Escherichia coli and Klebsiella spp. Concordant ESBL‐PE species were recovered from rectal screening and clinical samples in 7.46% (n = 5/67) of foals. Conclusions and Clinical Importance On‐admission ESBL‐PE rectal colonization was associated with ESBL‐PE HAI in neonatal foals. The ESBL‐PE infections were associated with surgery during hospitalization. These findings emphasize the importance of optimal infection control and treatment of clinical infections in equine neonatal intensive care units.

Rabies is a fatal zoonotic disease affecting all mammalian species. It is caused by the rabies virus and is prevalent worldwide. Horses are not commonly infected with rabies but their vaccination is recommended due to the potential zoonotic risk. This study aimed to evaluate the duration of immunity following rabies vaccination in horses. A total of 126 serum samples were collected from 93 horses, vaccinated 6 to 91 months before sampling. Rabies-virus-neutralizing antibody (RVNA) levels were evaluated using the Rabies Fluorescent Focus Inhibition Test (RFFIT). A protective RVNA titer of above 0.5 IU/mL was found in 112 (88.9%) of the samples and 84 (90.3%) of the horses. Antibody titers declined over time (rho = −0.271, p = 0.002); however, there was no significant difference in antibody titers or the prevalence of unprotected horses between the time intervals following vaccination. Purebred horses had lower antibody titers (p = 0.024). The response to booster vaccination was inspected in ten horses, and increased antibody titers were found in eight of them. The results of this study demonstrate the prolonged persistence of protective immunity in horses following rabies vaccination, in some cases, for up to eight years. Therefore, the current annual vaccination strategy should be re-evaluated. A rate of 9.7% of poor responders should be considered from an epidemiological perspective in order to minimize the risk of emergence of the disease.

An interrupted time-series study design was implemented to evaluate the impact of antibiotic stewardship interventions on antibiotic prescribing among veterinarians. A total of 41 veterinarians were enrolled in Canada and Israel and their prescribing data between 2019 and 2021 were obtained. As an intervention, veterinarians periodically received three feedback reports comprising feedback on the participants' antibiotic prescribing and prescribing guidelines. A change in the level and trend of antibiotic prescribing after the administration of the intervention was compared using a multi-level generalized linear mixed-effect negative-binomial model. After the receipt of the first (incidence rate ratios [IRR] = 0.88; 95% confidence interval (CI): 0.79, 0.98), and second (IRR = 0.85; 95% CI: 0.75, 0.97) feedback reports, there was a reduced prescribing rate of total antibiotic when other parameters were held constant. This decline was more pronounced among Israeli veterinarians compared to Canadian veterinarians. When other parameters were held constant, the prescribing of critical antibiotics by Canadian veterinarians decreased by a factor of 0.39 compared to that of Israeli veterinarians. Evidently, antibiotic stewardship interventions can improve antibiotic prescribing in a veterinary setting. The strategy to sustain the effect of feedback reports and the determinants of differences between the two cohorts should be further explored.

Abstract Background Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. Methods Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer’s instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. Results The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test’s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. Conclusions The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection. Graphical Abstract

West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.

Besnoitia is a tissue cyst forming coccidia, which affects multiple host species worldwide. Equine besnoitiosis is characterized mainly by generalized skin lesions and cysts in the scleral conjunctiva. Recent reports revealed exposure to Besnoitia in equines in Europe and the United States. However, the exposure to Besnoitia spp. in the Israeli equine population was never investigated. The aim of this study was to evaluate the seroprevalence and associated risk factors for besnoitiosis in equids in Israel. A cross-sectional serosurvey was performed using serum samples of apparently healthy horses (n = 347), donkeys (n = 98), and mules (n = 6), and exposure to Besnoitia spp. was determined by an immunofluorescent antibody test (IFAT). Anti-Besnoitia spp. antibodies were detected in 17.7% equids, 6.9% horses, 33.3% mules, and 55.1% donkeys. The seroprevalence in donkeys was significantly higher than in horses (p < 0.001). A significant association between the geographic location and seropositivity was found both in horses and donkeys, which was significantly higher (p = 0.004) in horses sampled in southern Israel, and donkeys sampled in Israel versus the Palestinian Authority (p < 0.001). This is the first serosurvey of Besnoitia infection in equines in Israel, and the results are consistent with reports from Europe. The clinical significance of equine besnoitiosis should be further investigated.

SummaryBackgroundPulmonary oedema is an uncommon but life‐threatening condition in horses. It is defined as an abnormal accumulation of liquid and solute in the extravascular tissues and spaces of the lungs, which causes oxygen diffusion impairment and a reduction in lung compliance. Diagnosis is determined by typical clinical signs, including frothy, haemorrhagic nasal discharge and respiratory distress, and can be supported by thoracic imaging and arterial blood gas analysis. In horses, post‐anaesthetic pulmonary oedema (PAPOE) is poorly reported, and there is a lack of information regarding risk factors and prognosis.ObjectivesThe objective of the study was to determine risk factors and short‐term outcome for PAPOE during the recovery period in horses.Study designA retrospective case‐control study including 17 cases and 53 controls.MethodsMedical records of clinical cases and controls over a period of four and a half years were reviewed. Short‐term outcomes and risk factors for PAPOE were investigated using conditional multivariable logistic regression analysis.ResultsAscarid impaction prevalence in PAPOE cases was significantly higher than controls (p = 0.03). In a univariable analysis, the following variables were significantly associated with PAPOE (p ≤ 0.05): gastric reflux on admission, haemorrhage during surgery, intravenous crystalloid treatment prior to admission, fresh frozen plasma and hydroxyethyl starch administration during anaesthesia. On multivariable analysis, gastric reflux on admission (OR = 7.9, 95% CI 1.1, 57.2) and fresh frozen plasma treatment during anaesthesia (OR = 9.6, 95% CI 1.2, 79.1) were found as risk factors for PAPOE. There was no significant difference in outcome between cases and controls.Main limitationsThe low number of cases, the lack of gross and histological post‐mortem examinations of the horses that did not survive and the clinical definition of PAPOE.ConclusionsGastric reflux and fresh frozen plasma administration during general anaesthesia are associated with PAPOE.

In order to evaluate the contribution of different wild bird species to West Nile virus (WNV) circulation in Israel, during the months preceding the 2018 outbreak that occurred in Israel, we randomly sampled 136 frozen carcasses of a variety of avian species. Visceral and central nervous system (CNS) tissue pools were tested using WNV NS2A RT qPCR assay; of those, 15 (11.03%, 95% CI: 6.31–17.54%) tissue pools were positive. A total of 13 out of 15 WNV RT qPCR positive samples were successfully sequenced. Phylogenetic analysis indicated that all WNV isolates were identified as lineage 1 and all categorized as cluster 2 eastern European. Our results indicated that WNV isolates that circulated within the surveyed wild birds in spring 2018 were closely related to several of the isolates of the previously reported 2018 outbreak in birds in Israel and that the majority of infected birds were of local species.

Antibiotic stewardship interventions such as prescribing guidelines and periodic feedback on antibiotic prescribing, can positively influence prescribing behaviour among physicians. Similarly, the volume and type of antibiotics prescribed in veterinary settings could be improved by implementing such interventions. An interrupted time-series study design was implemented to evaluate the impact on antibiotics prescribing rate among veterinarians, upon receipt of antibiotic stewardship interventions generated by a web-based platform (OPEN Stewardship).A total of 41 veterinarians were enrolled in Canada and Israel and their antibiotic prescribing data between 2019 and 2021 were obtained. After a brief pre-intervention period, veterinarians periodically received three OPEN Stewardship generated interventions in the form of feedback reports that provided relevant prescribing guidelines and feedback on the participants’ antibiotic prescribing. A multi-level generalized linear mixed-effect negative binomial model was used to compare the change in the level and the trend of antibiotic prescribing after the administration of the intervention.A decline in antibiotic prescribing rate was found among the Canadian and the Israeli veterinarians who received the feedback reports. After the receipt of the first (incidence rate ratios [IRR] = 0·88; 95% confidence interval: 0·79, 0·98), and second (IRR = 0·85; 95% confidence interval: 0·75, 0·97) feedback reports, there was a reduced prescribing rate of total antibiotic when other parameters were held constant. In general, after the receipt of the feedback reports, decline in antibiotics prescribing was more pronounced among Israeli veterinarians compared to Canadian veterinarians. When other parameters were held constant, following the receipt of feedback reports, the prescribing of critical antibiotics by Canadian veterinarians decreased by a factor of 0·39 compared to that of Israeli veterinarians.This study shows that antibiotic prescribing can be improved in a veterinary setting by antibiotic stewardship interventions such as feedback on antibiotic prescribing and prescribing guidelines. The strategy to sustain the effect of feedback reports and the determinants of differences between the two cohorts should be further explored.Funding Information: This work was supported by the funding from the Canadian Institutes of Health Research (CIHR) (grant # AMR352 155212) and the Israeli Ministry of Health (grant #8762491), under the European Commission's Joint Programming Initiative on Antimicrobial Resistance (5th Joint Call).Declaration of Interests: The authors have no conflict of interest to declare.Ethics Approval Statement: This study was approved by research ethics boards in both Canada (University of Guelph REB #19-03-006), and Israel (Internal Research Review Committee of the Koret School of Veterinary Medicine – Veterinary Teaching Hospital #KSVM-VTH/05_2019).

Equine encephalosis (EE) is an arthropod-borne, noncontagious, febrile disease of horses. It is caused by EE virus (EEV), an Orbivirus of the Reoviridae family transmitted by Culicoides. Within the EEV serogroup, seven serotypes (EEV-1–7) have been identified to date. This virus was first isolated from a horse in South Africa in 1967 and until 2008 was believed to be restricted to southern Africa. In 2008–2009, isolation of EEV in an outbreak reported from Israel demonstrated the emergence of this pathogen into new niches. Indeed, testing in retrospect sera samples revealed that EEV had already been circulating outside of South Africa since 2001. Although EEV normally does not cause severe clinical disease, it should be considered important since it may indicate the possible spread of other related, much more pathogenic viruses, such as African horse sickness virus (AHSV). The spread of EEV from South Africa to central Africa, the Middle East and India is an example of the possible emergence of new pathogens in new niches, as was seen in the case of West Nile virus, and should be a reminder not to limit the differential list when facing a possible outbreak or a cluster of clinical cases. This review summarizes current knowledge regarding EEV structure, pathogenesis, clinical significance, and epidemiology.

Background: antimicrobial resistance is a global problem in human and veterinary medicine. We aimed to investigate the extended spectrum β-lactamase-producing Enterobacterales (ESBL-PE) gut colonization in healthy community dogs in Israel. Methods: Rectal swabs were sampled from 145 healthy dogs, enriched, plated on selective plates, sub-cultured to obtain pure cultures, and ESBL production was confirmed. Bacterial species and antibiotic susceptibility profiles were identified. WGS was performed on all of the ESBL-PE isolates and their resistomes were identified in silico. Owners’ questionnaires were collected for risk factor analysis. Results: ESBL-PE gut colonization rate was 6.2% (n = 9/145, 95% CI 2.9–11.5). Overall, ten isolates were detected (one dog had two isolates); the main species was Escherichia coli (eight isolates), belonging to diverse phylogenetic groups—B1, A and C. Two isolates were identified as Citrobacter braakii, and C. portucalensis. A phylogenetic analysis indicated that all of the isolates were genetically unrelated and sporadic. The isolates possessed diverse ESBL genes and antibiotic-resistance gene content, suggesting independent ESBL spread. In a multivariable risk factor analysis, coprophagia was identified as a risk factor for ESBL-PE gut colonization (p = 0.048, aOR = 4.408, 95% CI 1.014–19.169). Conclusions: healthy community dogs may be colonized with ESBL-PE MDR strains, some of which were previously reported in humans, that carry wide and diverse resistomes and may serve as a possible source for AMR.

Extended-spectrum β-lactamases (ESBLs) are a growing public health threat, and one key human exposure point is through livestock and the food supply. Understanding microbiome factors associated with fecal ESBL carriage can help detect and ideally assist with controlling and preventing ESBL dissemination among livestock. The objective of this study was to investigate the diversity and composition of the heifer fecal microbiota in ESBL-producing Enterobacterales (ESBL-PE) carriers and noncarriers. A total of 59 fecal samples were collected from replacement heifers between 12 and 18 months old from eight dairy farms in central Israel. Genomic DNA was extracted, and 16S rRNA amplicon sequencing was performed (Illumina short reads), focusing on a comparison between 33 ESBL-PE carriers (55.9%) and 26 (44.1%) noncarriers. Samples were analyzed and compared using QIIME2 (DADA2 pipeline and taxonomic assignment with SILVA database) and associated R packages for alpha and beta diversity and taxonomic abundances. Alpha diversity (Shannon diversity) and beta diversity (unweighted UniFrac) showed no significant difference between ESBL-PE carriers and noncarriers. Heifers from farms feeding calves with pooled colostrum had higher ESBL-PE carriage rates than heifers from farms feeding with individual mother colostrum (p < 0.001). Taxonomical abundance analysis revealed that the most common bacterial phyla were Bacteroidetes (44%) and Firmicutes (38%). There was no significant difference in taxonomic composition between ESBL-PE carriers and noncarriers at the phylum and genus levels. However, LEfSe biomarker discovery analysis identified several genera which were significantly different between carriers and noncarriers. For example, Prevotellacaea, Bacteroides, Rikenellaceae, and uncultured Bacteroidales were more abundant in ESBL carriers than noncarriers. Some aspects of microbiota composition differ between ESBL carriers and noncarriers in dairy heifers, specifically the abundance of certain genera. Feeding with pooled colostrum may play a role in that assembly. These could potentially serve as markers of ESBL-PE carriage. However, further research is needed to determine whether these observed differences have a significant impact on colonization with ESBL-PE.

The relationship between men and horses has significantly evolved over the last century [...].

Equine Neospora infection has been linked to neurological disorders and infertility in horses. This study looked into the risk factors for infection and the exposure to Neospora spp. in horses. The study was performed in two independent populations in Israel. The first consisted of apparently healthy horses, and the second consisted of mares examined during pregnancy and after parturition. Sera samples collected from horses and mares were tested for Neospora exposure by the indirect fluorescent antibody test (IFAT). The study revealed seroprevalence of 24% in apparently healthy horses and 66.4% and 48.6% in mares during gestation and after parturition, respectively. Among the investigated risk factors, older age (p = 0.026) and housing in both stalls and paddocks (p = 0.033) in apparently healthy horses, and Arabian breeds (p = 0.005) in pregnant mares, were found to be significantly associated with Neospora spp. seropositivity in univariable, but not multivariable, statistical analysis. This study revealed high exposure of equines to Neospora parasites, especially mares. Horse farm management, in combination with active surveillance, including serological testing and follow up, could help reduce the spread of the parasite among horses in endemic areas.

Equine piroplasmosis (EP) is a widely spread tick-borne disease of horses, caused by the hemoparasites Theileria equi and Babesia caballi. Although most horses in endemic areas are subclinical carriers of parasites, acute or peracute disease may occur, especially when naive horses are infected. This case report presents a characteristic case of peracute and fatal EP in a yearling filly, that was transferred from stable to pasture with first exposure to ticks. The filly was diagnosed with high parasitemia of both T. equi and B. caballi, with co-infection of both parasites within the same erythrocyte. Post mortem findings revealed multi-organ damage (spleen, kidney, lungs and muscles) and systemic bleeding. Quantitative PCR revealed high parasitemia of both parasites, consistent with acute and clinical infection in the sick filly. An epidemiological investigation including molecular analyses of blood samples was conducted on the farm, in which of thirteen horses, two were carriers of T. equi and one was a carrier of B. caballi with similar genotypes to the filly. The described case emphasizes the importance of implementation of preventive measures when transferring naive horses to tick infected pasture in endemic areas. In addition, identifying the previously undocumented presence of co-infection within the same erythrocytes raises questions regarding the pathogenesis and cell invasion mechanisms of these parasites, with possible future therapeutic implications.

Babesia caballi is a tick-borne hemoparasite of equines and one of the causative agents of equine piroplasmosis, which poses a great concern for the equine industry regarding animal welfare and international horse movement. The parasite is endemic in Israel; however, its seroprevalence in the area was never evaluated due to antigenic heterogenicity in the gene used in the commercially available kit. Blood samples were collected from 257 horses at 19 farms throughout the country and screened for the presence of anti-B. caballi antibodies via an indirect immunofluorescent antibody test (IFAT) and for the presence of parasite DNA by nested PCR. The seroprevalence of B. caballi was 69.6% and its molecular prevalence was 9.7%. The geographical area, horse’s sex, breed, housing, exposure to ticks, and specifically to Hyalomma marginatum, and co-infection with Theileria equi were found to be significantly associated with serologic exposure in univariable analysis, while the geographical area and horses’ sex remained significant in the multivariable analysis. The results of this study demonstrate a high level of exposure to B. caballi and identify important risk factors for infection. The difference between the serological and molecular prevalence, probably related to parasite clearance, is also highlighted.

Introduction Antimicrobial resistance (AMR) impacts the health and well-being of animals, affects animal owners both socially and economically, and contributes to AMR at the human and environmental interface. The overuse and/or inappropriate use of antibiotics in animals has been identified as one of the most important drivers of the development of AMR in animals. Effective antibiotic stewardship interventions such as feedback can be adopted in veterinary practices to improve antibiotic prescribing. However, the provision of dedicated financial and technical resources to implement such systems are challenging. The newly developed web-based Online Platform for Expanding Antibiotic Stewardship (OPEN Stewardship) platform aims to automate the generation of feedback reports and facilitate wider adoption of antibiotic stewardship. This paper describes a protocol to evaluate the usability and usefulness of a feedback intervention among veterinarians and assess its impact on individual antibiotic prescribing. Methods and analysis Approximately 80 veterinarians from Ontario, Canada and 60 veterinarians from Israel will be voluntarily enrolled in a controlled interrupted time-series study and their monthly antibiotic prescribing data accessed. The study intervention consists of targeted feedback reports generated using the OPEN Stewardship platform. After a 3-month preintervention period, a cohort of veterinarians (treatment cohort, n=120) will receive three feedback reports over the course of 6 months while the remainder of the veterinarians (n=20) will be the control cohort. A survey will be administered among the treatment cohort after each feedback cycle to assess the usability and usefulness of various elements of the feedback report. A multilevel negative-binomial regression analysis of the preintervention and postintervention antibiotic prescribing of the treatment cohort will be performed to evaluate the impact of the intervention. Ethics and dissemination Research ethics board approval was obtained at each participating site prior to the recruitment of the veterinarians. The study findings will be disseminated through open-access scientific publications, stakeholder networks and national/international meetings.

In human medicine, infections caused by third-generation cephalosporin-resistant Enterobacterales (3GCRE) are associated with detrimental outcomes. In veterinary medicine, controlled epidemiological analyses are lacking. A matched case–case–control investigation (1:1:1 ratio) was conducted in a large veterinary hospital (2017–2019). In total, 29 infected horses and donkeys were matched to 29 animals with third-generation cephalosporin-susceptible Enterobacterales (3GCSE) infections, and 29 uninfected controls (overall n = 87). Despite multiple significant associations per bivariable analyses, the only independent predictor for 3GCRE infection was recent exposure to antibiotics (adjusted odds ratio (aOR) = 104, p < 0.001), but this was also an independent predictor for 3GCSE infection (aOR = 22, p < 0.001), though the correlation with 3GCRE was significantly stronger (aOR = 9.3, p = 0.04). In separated multivariable outcome models, 3GCRE infections were independently associated with reduced clinical cure rates (aOR = 6.84, p = 0.003) and with 90 days mortality (aOR = 3.6, p = 0.003). Klebsiella spp. were the most common 3GCRE (36%), and blaCTX-M-1 was the major β-lactamase (79%). Polyclonality and multiple sequence types were evident among all Enterobacterales (e.g., Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae). The study substantiates the significance of 3GCRE infections in equine medicine, and their independent detrimental impact on cure rates and mortality. Multiple Enterobacterales genera, subtypes, clones and mechanisms of resistance are prevalent among horses and donkeys with 3GCRE infections.