The effect of systemic acetazolamide administration on intraocular pressure in healthy horses-A preliminary study.

OBJECTIVES: In equine glaucoma, topical treatment with carbonic anhydrase inhibitors (CAIs) is recommended. Oral acetazolamide, a systemic CAI, is used in horses with hyperkalemic periodic paralysis. Information regarding its effect on equine intraocular pressure (IOP) is scarce. The aim of the study was to determine the effect of oral acetazolamide treatment on IOP in horses, in a case-control study. ANIMALS: Ten healthy horses. PROCEDURES: Horses were treated with oral acetazolamide (4.4 mg/kg) BID for 1 week. Serum acetazolamide concentrations were determined by liquid chromatography/tandem mass spectrometry, and IOP were measured before treatment, daily during treatment, and at 48 and 72 h after treatment. RESULTS: Acetazolamide serum levels reached steady state at 72 h after the first oral dose. In a mixed effect model logistic regression, there was a significant decrease in IOP on the third treatment day, of 2.4 mmHg (p = .012) and 2.7 mmHg (p = .006) in the left (OS) and right eye (OD), respectively. On the seventh day, there was a decrease in 2.5 mmHg (p = .008) and 2.7 mmHg (p = .007) OS and OD, respectively. A significant increase occurred 48 h following treatment discontinuation (3.6 mmHg, p < .001 and 3.5 mmHg, p < .001 OS and OD, respectively). The area under the concentration versus time curve (AUC(0-10h)) was 1.1 ± 0.5 µg/mL*h, mean residence time 6.7 ± 4.3 h, peak plasma concentration (Cmax) 0.4 ± 0.4 µg/mL and time to reach Cmax 1.8 h. There was a significant increase in serum concentrations 1, 2, 48, 72, and 156 h following the first drug administration (p < .05). CONCLUSIONS: Further studies are required to determine whether acetazolamide is a potential treatment for equine glaucoma.

Prevalence and Molecular Characterization of Extended-Spectrum ß-Lactamase Producing Enterobacterales in Healthy Community Dogs in Israel.

BACKGROUND: antimicrobial resistance is a global problem in human and veterinary medicine. We aimed to investigate the extended spectrum ß-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.

Seroprevalence of Leptospira spp. in Horses in Israel.

Leptospirosis has been reported in both humans and animals in Israel but has not been reported in horses. In 2018, an outbreak of Leptospira spp. serogroup Pomona was reported in humans and cattle in Israel. In horses, leptospirosis may cause equine recurrent uveitis (ERU). This report describes the first identification of Leptospira serogroup Pomona as the probable cause of ERU in horses in Israel, followed by an epidemiological investigation of equine exposure in the area. Serologic exposure to Leptospira was determined by microscopic agglutination test (MAT) using eight serovars. In 2017, serovar Pomona was identified in a mare with signs of ERU. Seven of thirteen horses from that farm were seropositive for serogroup Pomona, of which three had signs of ERU. During the same time period, 14/70 horses from three other farms were positive for serogroup Pomona. In 2015, two years prior to this diagnosis, 259 horses from 21 farms were sampled and one horse tested seropositive for serovar Icterohaemorrhagiae. In 2018, one year later, 337 horses were sampled on 29 farms, with none testing seropositive. Although horses are not considered a major host of Leptospira spp., it appears that horses may be infected, and clinically affected, in the course of an outbreak in other species. The identification of leptospirosis in stabled horses may impose a significant zoonotic risk to people.

Third Generation Cephalosporin Resistant Enterobacterales Infections in Hospitalized Horses and Donkeys: A Case-Case-Control Analysis.

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 ß-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.

Extended spectrum ß lactamase-producing Enterobacteriaceae shedding by race horses in Ontario, Canada.

BACKGROUND: We aimed to investigate the prevalence, molecular epidemiology and prevalence factors for Extended Spectrum ß-Lactamase-producing Enterobacteriaceae (ESBL-E) shedding by race horses. A cross-sectional study was performed involving fecal samples collected from 169 Thoroughbred horses that were housed at a large racing facility in Ontario, Canada. Samples were enriched, plated on selective plates, sub-cultured to obtain pure cultures and ESBL production was confirmed. Bacterial species were identified and antibiotic susceptibility profiles were assessed. E. coli sequence types (ST) and ESBL genes were determined using multilocus sequence type (MLST) and sequencing. Whole genome sequencing was performed to isolates harboring CTX-M-1 gene. Medical records were reviewed and associations were investigated. RESULTS: Adult horses (n = 169), originating from 16 different barns, were sampled. ESBL-E shedding rate was 12% (n = 21/169, 95% CI 8-18%); 22 ESBL-E isolates were molecularly studied (one horse had two isolates). The main species was E. coli (91%) and the major ESBL gene was CTX-M-1 (54.5%). Ten different E. coli STs were identified. Sixty-four percent of total isolates were defined as multi-drug resistant. ESBL-E shedding horses originated from 8/16 different barns; whereas 48% (10/21) of them originated from one specific barn. Overall, antibiotic treatment in the previous month was found as a prevalence factor for ESBL-E shedding (p = 0.016, prevalence OR = 27.72, 95% CI 1.845-416.555). CONCLUSIONS: Our findings demonstrate the potential diverse reservoir of ESBL-E in Thoroughbred race horses. Multi-drug resistant bacteria should be further investigated to improve antibiotic treatment regimens and equine welfare.

Extended-Spectrum ß-Lactamase-Producing Enterobacterales Shedding by Dogs and Cats Hospitalized in an Emergency and Critical Care Department of a Veterinary Teaching Hospital.

Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) gut shedding in human medicine is considered as a major reservoir for ESBL-associated infections in high risk patients. In veterinary medicine, data regarding ESBL-PE gut shedding on admission to emergency and critical care department is scarce. We aimed to determine ESBL-PE shedding rates by dogs and cats in this setting and to determine the risk factors for shedding, at two separate periods, three-years apart. Rectal swabs were collected from animals, on admission and 72 h post admission, enriched and plated on Chromagar ESBL plates, followed by bacterial identification. ESBL phenotype was confirmed and antibiotic susceptibility profiles were determined (Vitek 2). Medical records were reviewed for risk factor analysis (SPSS). Overall, 248 animals were sampled, including 108 animals on period I (2015-2016) and 140 animals on period II (2019). In both periods combined, 21.4% of animals shed ESBL-PE on admission, and shedding rates increased significantly during hospitalization (53.7%, p-value < 0.001). The main ESBL-PE species were Escherichia coli and Klebsiella pneumoniae, accounting for more than 85% of the isolates. In a multivariable analysis, previous hospitalization was a risk factor for ESBL-PE gut shedding (p-value = 0.01, Odds ratio = 3.05, 95% Confidence interval 1.28-7.27). Our findings demonstrate significant ESBL-PE gut shedding among small animals in the emergency and critical care department, posing the necessity to design and implement control measures to prevent transmission and optimize antibiotic therapy in this setting.

Genomic Characterization of Antimicrobial Resistance, Virulence, and Phylogeny of the Genus Ochrobactrum.

Ochrobactrum is a ubiquitous Gram-negative microorganism, mostly found in the environment, which can cause opportunistic infections in humans. It is almost uniformly resistant to penicillins and cephalosporins through an AmpC-like ß-lactamase enzyme class (OCH). We studied 130 assembled genomes, of which 5 were animal-derived isolates recovered in Israel, and 125 publicly available genomes. Our analysis focused on antimicrobial resistance (AMR) genes, virulence genes, and whole-genome phylogeny. We found that 76% of Ochrobactrum genomes harbored a blaOCH ß-lactamase gene variant, while 7% harbored another AmpC-like gene. No virulence genes other than lipopolysaccharide-associated genes were found. Core genome multilocus sequence typing clustered most samples to known species, but neither geographical clustering nor isolation source clustering were evident. When analyzing the distribution of different blaOCH variants as well as of the blaOCH-deficient samples, a clear phylogenomic clustering was apparent for specific species. The current analysis of the largest collection to date of Ochrobactrum genomes sheds light on the resistome, virulome, phylogeny, and species classification of this increasingly reported human pathogen. Our findings also suggest that Ochrobactrum deserves further characterization to underpin its evolution, taxonomy, and antimicrobial resistance.

Extended-Spectrum ß-lactamase-Producing Enterobacteriaceae Shedding in Farm Horses Versus Hospitalized Horses: Prevalence and Risk Factors.

: We aimed to investigate the prevalence, molecular characteristics and risk factors of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) shedding in horses. A prospective study included three cohorts: (i) farm horses (13 farms, n = 192); (ii) on hospital admission (n = 168) and; (iii) horses hospitalized for ≥72 h re-sampled from cohort (ii) (n = 86). Enriched rectal swabs were plated, ESBL-production was confirmed (Clinical and Laboratory Standards Institute (CLSI)) and genes were identified (polymerase chain reaction (PCR)). Identification and antibiotic susceptibility were determined (Vitek-2). Medical records and owners' questionnaires were analyzed. Shedding rates increased from 19.6% (n = 33/168) on admission to 77.9% (n = 67/86) during hospitalization (p < 0.0001, odds ratio (OR) = 12.12). Shedding rate in farms was 20.8% (n = 40/192), significantly lower compared to hospitalized horses (p < 0.0001). The main ESBL-E species (n = 192 isolates) were E. coli (59.9%, 115/192), Enterobacter sp. (17.7%, 34/192) and Klebsiella pneumoniae (13.0%, 25/192). The main gene group was CTX-M-1 (56.8%). A significant increase in resistance rates to chloramphenicol, enrofloxacin, gentamicin, nitrofurantoin, and trimethoprim-sulpha was identified during hospitalization. Risk factors for shedding in farms included breed (Arabian, OR = 3.9), sex (stallion, OR = 3.4), and antibiotic treatment (OR = 9.8). Older age was identified as a protective factor (OR = 0.88). We demonstrated an ESBL-E reservoir in equine cohorts, with a significant ESBL-E acquisition, which increases the necessity to implement active surveillance and antibiotic stewardship programs.

CTX-M-15 Producing Escherichia coli Sequence Type 361 and Sequence Type 38 Causing Bacteremia and Umbilical Infection in a Neonate Foal.

An eighteen-hour-old Tennessee walking horse foal was referred due to weakness and abdominal pain. Physical examination revealed dehydration, distended abdomen, and uveitis. Blood analysis revealed leukopenia, neutrophils' toxicity and left shift. The foal developed bloody diarrhea, gastric reflux, and was diagnosed with sepsis and enterocolitis. The foal was treated with intravenous fluids, plasma, antibiotics (ceftriaxone and metronidazole), partial parenteral nutrition (dextrose and amino acids), flunixin meglumine, and ophthalmic drops. Umbilical ultrasound revealed a fluid pocket adjacent to the umbilical vein; therefore, omphalectomy was performed. Umbilicus and blood were cultured. Results recovered two multidrug-resistant extended-spectrum ß-lactamase (ESBL) producing Escherichia coli clones, identified as ST38 (umbilicus) and ST361 (blood), harboring two different plasmids encoding blaCTX-M-15. Antibiotic treatment was replaced with imipenem and amikacin, but the foal deteriorated and was euthanized. Postmortem investigation revealed severe ulcerative enteritis, a perforation site and acute renal infarcts. Sepsis due to several different ESBL-producing E. coli strains should be considered, investigated, and treated accordingly.

Emergence and Spread of Different ESBL-Producing Salmonella enterica Serovars in Hospitalized Horses Sharing a Highly Transferable IncM2 CTX-M-3-Encoding Plasmid.

Salmonella enterica is a major causative pathogen of human and animal gastroenteritis. Antibiotic resistant strains have emerged due to the production of extended-spectrum ß-lactamases (ESBLs) posing a major health concern. With the increasing reports on ESBL-producing Enterobacterales that colonize companion animals, we aimed to investigate ESBL dissemination among ESBL-producing Salmonella enterica (ESBL-S) in hospitalized horses. We prospectively collected ESBL-S isolates from hospitalized horses in a Veterinary-Teaching Hospital during Dec 2015-Dec 2017. Selection criteria for ESBL-S were white colonies on CHROMagarESBL plates and an ESBL phenotypic confirmation. Salmonella enterica serovars were determined using the Kaufmann-White-Le-Minor serological scheme. ESBL-encoding plasmids were purified, transformed and compared using restriction fragment length polymorphism (RFLP). Whole genome sequencing (Illumina and MinION platforms) were performed for detailed phylogenetic and plasmid analyses. Twelve ESBL-S were included in this study. Molecular investigation and Sequence Read Archive (SRA) meta-analysis revealed the presence of three unique Salmonella enterica serovars, Cerro, Havana and Liverpool, all reported for the first time in horses. PFGE revealed the clonal spread of S. Cerro between seven horses. All twelve isolates carried bla CTX-M- 3 and showed an identical multidrug resistance profile with co-resistance to trimethoprim/sulfamethoxazole and to aminoglycosides. Plasmid RFLP proved the inter-serovar horizontal spread of a single bla CTX-M- 3-encoding plasmid. Complete sequence of a representative plasmid (S. Havana strain 373.3.1), designated pSEIL-3 was a -86.4 Kb IncM2 plasmid, that encoded nine antibiotic resistance genes. pSEIL-3 was virtually identical to pCTX-M3 from Citrobacter freundii, and showed high identity (>95%) to six other bla CTX-M- 3 or bla NDM- 1 IncM2 broad host range plasmids from various Enterobacterales of human origin. Using a specific six gene-based multiplex PCR, we detected pSEIL-3 in various Enterobacterales species that co-colonized the horses' gut. Together, our findings show the alarming emergence of ESBL-S in hospitalized horses associated with gut shedding and foal morbidity and mortality. We demonstrated the dissemination of CTX-M-3 ESBL among different Salmonella enterica serovars due to transmission of a broad host range plasmid. This report highlights horses as a zoonotic reservoir for ESBL-S, including highly transmissible plasmids that may represent a 'One-Health' hazard. This risk calls for the implementation of infection control measures to monitor and control the spread of ESBL-S in hospitalized horses.

Petting Zoo Animals as an Emerging Reservoir of Extended-Spectrum ß-Lactamase and AmpC-Producing Enterobacteriaceae.

Extended spectrum beta-lactamases and AmpC-producing Enterobacteriaceae (ESBL/AmpC-E) have become a great concern in both human and veterinary medicine. One setting in which this risk could be particularly prominent is petting zoos, in which humans, especially children, directly and indirectly interact with the animals. Yet, while the zoonotic transmission of various Enterobacteriaceae has been reported previously in petting zoos, reports on ESBL/AmpC-E shedding in this setting is currently lacking, despite the high potential risk. To fill this knowledge gap, we conducted a prospective cross-sectional study to explore the prevalence, molecular epidemiology, and risk for shedding of ESBL/AmpC-E in petting zoos. We performed a prospective cross-sectional study in eight petting zoos. Altogether, we collected 381 fecal and body-surface samples from 228 animals, broth-enriched them, and then plated them onto CHROMagar ESBL-plates for ESBL/AmpC-E isolation. Next, we identified the isolated species and tested their susceptibility to various antibiotics using the Vitek-2 system, determined bacterial relatedness by multilocus sequence typing (MLST), and identified ESBL/AmpC genes by using PCR and sequencing. Finally, we asked petting zoo owners and veterinarians to complete questionnaires, which we then analyzed to evaluate risk factors for ESBL/AmpC-E shedding. We found that ESBL/AmpC-E shedding is an important, currently oversighted risk in petting zoos, as the overall shedding rate was 12% (35 isolates, including 29% ESBL-producers, 34% AmpC-producers, and 37% ESBL and AmpC-producers). The isolated bacteria included Enterobacter cloacae (55%), Escherichia coli (31%), and Citrobacter freundii (14%), with diverse ESBL genes. MLST revealed diverse sequence types (STs), including the highly virulent Enterotoxigenic ST656 and the Uropathogenic ST127 E. coli strains, indicating complex epidemiology with inter-animal bacterial transmission. Shedding was associated with petting permission and antibiotic treatment in the petting zoo (OR = 7.34), which were identified as risk factors for ESBL/AmpC shedding. Our findings highlight petting zoos as a source for antibiotic-resistant ESBL/AmpC-producing bacteria, including highly virulent, disease-associated MDR E. coli strains. As this risk has not been previously described in detail, it calls for the implementation of infection control and active surveillance programs in petting zoos and raises the need for a comprehensive guideline to restrain this emerging concern.

Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae in Hospitalized Neonatal Foals: Prevalence, Risk Factors for Shedding and Association with Infection.

Extended-spectrum ß-lactamase Enterobacteriaceae (ESBL-E) have been investigated in adult horses, but not in foals. We aimed to determine shedding and infection in neonatal foals and mares. Rectal swabs were sampled from mare and foal pairs on admission and on the 3rd day of hospitalization; enriched, plated, and bacteria were verified for ESBL production. Identification and antibiotic susceptibility profiles were determined (Vitek2). Genotyping was performed by multilocus sequence typing (MLST). Genes were identified by PCR and Sanger sequencing. Medical data were analyzed for risk factors (SPSS). On admission, 55 pairs were sampled, of which 33 pairs were re-sampled. Shedding rates on admission in foals and mares were 33% (95% CI 21-47%) and 16% (95% CI 8-29%), respectively, and during hospitalization, these increased significantly to 85% (95% CI 70-94%) and 58% (95% CI 40-73%), respectively. Foal shedding was associated with umbilical infection on admission (P = 0.016) and with ampicillin treatment during hospitalization (p = 0.011), and was independent of the mare's shedding. The most common ESBL-E was Escherichia coli. During hospitalization, species diversity increased. Four foals were infected with ESBL-E strains, including umbilical infections and wounds. This study substantiates an alarming prevalence of shedding in neonatal foals, which should be further investigated in order to reduce resistance rates.

Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys.

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE: The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.