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Fungal and bacterial culture is currently the primary method for pathogen detection and identification. Next-generation sequencing is a powerful method for detecting and identifying the presence of microbial DNA in samples. We evaluated the correlation between fungal and bacterial culture with next-generation sequencing in equine uterine samples. Fungal cultures (n=63) were evaluated based on their culture results. In culture positive (n=16) samples, next-generation sequencing identified the same organism in 10 samples (62.5%), 5 samples did not identify fungal agents, and 1 sample identified other species of fungal agents. In no growth samples (n=42), next-generation sequencing did not identify fungal agents in 37 samples (88.1%), 4 samples had a potential fungal pathogen identified, and one sample identified only non-pathogenic fungal organisms. Fungal culture and next-generation sequencing had an 80% agreement and moderate correlation by Kappa coefficient (0.508). Bacterial culture (n=57) was also evaluated based on bacterial culture results. In bacterial culture Positive samples (n=32), next-generation sequencing identified the same organism in 25 (78.1%) samples, and identified different organisms from the remaining 7 samples. In bacterial no growth samples (n=14), next-generation sequencing detected bacterial presence in 5 samples, and 9 samples had no bacterial DNA identified. The agreement between bacterial culture and next-generation sequencing was 74% with a moderate correlation by Kappa coefficient (0.46). In conclusion, culture and next-generation sequencing have moderate correlation, and next-generation sequencing has the potential as a diagnostic option for enhancing pathogen detection for equine endometritis., Competing Interests: Declaration of competing interest SHC received honoraria as a speaker at a veterinary conference from MicrogenVet., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: This report documents the exposure of passengers and crew of a commercial international flight to the zoonotic pathogen Brucella canis after an infected dog aborted in the passenger cabin of the aircraft. This case demonstrates the challenges associated with brucellosis screening and the risks that airline personnel, airport employees and travellers face when animals with unrecognized zoonotic infections are transported., Methods/results: The public health investigation of this case was conducted by the Centers for Disease Control, the Illinois Department of Health and the Illinois Department of Agriculture, in collaboration with a local veterinary clinic and several academic and federal diagnostic laboratories. It included an extensive diagnostic evaluation of the dam and aborted foetuses to confirm a diagnosis of canine brucellosis. Passengers, airline personnel and staff from the veterinary clinic where the dogs were treated underwent risk assessments, and clinic staff also received detailed guidance regarding infection prevention practices., Conclusions: Animal shelters and breeding programs are recommended to screen dogs routinely for brucellosis, but it is not unusual for domestic or imported animals to have unknown health histories, including the dog's brucellosis status, at the time of purchase, adoption, or re-homing. Testing recommendations and requirements vary by state, making it challenging for state public health and animal health agencies to monitor and respond appropriately. This case highlights the importance of Brucella spp. screening in sexually intact dogs prior to breeding, purchase, or domestic or international transportation of the dogs. The transportation of pregnant dogs may present a previously unrecognized public health threat in addition to contributing to unnecessary stress and health risks for pregnant animals., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Infections caused by antimicrobial-resistant Escherichia coli are the leading cause of death attributed to antimicrobial resistance (AMR) worldwide, and the known AMR mechanisms involve a range of functional proteins. Here, we employed a pan-genome wide association study (GWAS) approach on over 1,000 E. coli isolates from sick dogs collected across the US and Canada and identified a strong statistical association (empirical P < 0.01) of AMR, involving a range of antibiotics to a group 1 capsular (CPS) gene cluster. This cluster included genes under relaxed selection pressure, had several loci missing, and had pseudogenes for other key loci. Furthermore, this cluster is widespread in E. coli and Klebsiella clinical isolates across multiple host species. Earlier studies demonstrated that the octameric CPS polysaccharide export protein Wza can transmit macrolide antibiotics into the E. coli periplasm. We suggest that the CPS in question, and its highly divergent Wza, functions as an antibiotic trap, preventing antimicrobial penetration. We also highlight the high diversity of lineages circulating in dogs across all regions studied, the overlap with human lineages, and regional prevalence of resistance to multiple antimicrobial classes., Importance: Much of the human genomic epidemiology data available for E. coli mechanism discovery studies has been heavily biased toward shiga-toxin producing strains from humans and livestock. E. coli occupies many niches and produces a wide variety of other significant pathotypes, including some implicated in chronic disease. We hypothesized that since dogs tend to share similar strains with their owners and are treated with similar antibiotics, their pathogenic isolates will harbor unexplored AMR mechanisms of importance to humans as well as animals. By comparing over 1,000 genomes with in vitro antimicrobial susceptibility data from sick dogs across the US and Canada, we identified a strong multidrug resistance association with an operon that appears to have once conferred a type 1 capsule production system., Competing Interests: The authors declare no conflict of interest.

Non-aureus staphylococci and mammaliicocci (NASM) are the most frequently isolated bacterial group from bovine milk samples. Most studies focus on subclinical mastitis caused by NASM; however, NASM can cause clinical mastitis (CM) as well. We evaluated retrospective data from 6 years (2017-2022) to determine the species and frequency of NASM isolated from quarter bovine CM. The data was comprised of microbiological results from quarter CM samples routinely submitted to Quality Milk Production Services at Cornell University for microbial identification by MALDI-TOF mass spectrometry. A total of 9,909 microbiological results from 410 dairy herds were evaluated. Our results showed that 29 distinct NASM species were identified, with the 8 most prevalent NASM species being Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus simulans, Staphylococcus epidermidis, Staphylococcus sciuri (now Mammaliicoccus sciuri), Staphylococcus agnetis/Staphylococcus hyicus, Staphylococcus borealis, and Staphylococcus xylosus. The NASM distribution remained similar among seasons, but the frequency of NASM CM cases was higher during the summer. Our results showed different patterns of variations in the isolation frequency over time, depending on the bacterial species: increasing or decreasing trends, cyclic fluctuations, and, except for Staphylococcus borealis, a significant seasonality effect for our study's most prevalent NASM. This study showed that Staphylococcus chromogenes remains the most frequent (43%) NASM species identified from bovine CM, followed by Staphylococcus haemolyticus (18%), and Staphylococcus simulans (12%)., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Liquid handling is a fundamental capability for many scientific experiments. Previously, we introduced the Surface Patterned Omniphobic Tiles (SPOTs) platform, which enables manipulation of hundreds to thousands of independent experiments without costly equipment or excessive consumable expenses. However, the SPOTs platform requires a custom coating formulation and lacks robustness. To overcome these limitations, we introduce EZ-SPOTs. These devices can be created in an hour with common fabrication tools and just three components - glass, a hydrophobic coating, and acrylic. EZ-SPOTs preserve many of the SPOTs platform's strengths - ease of use, ability to handle a wide range of volumes, and scalability - and adopt a durable and abrasion resistant coating that enables multiple reuses of each device. Here, we describe the fabrication of EZ-SPOTs and showcase how its reusability allows antibiotic susceptibility testing of many isolates using a single device. These results quantitatively match current gold standard assays and the increased throughput provides substantially more information than standard approaches.

Objective: Brucella canis is a zoonotic bacterial pathogen of dogs that is notoriously difficult to diagnose and treat. Humans can become infected with B canis when an infected pet dog is brought into their home. Our objectives were to describe the clinical presentation and outcomes in dogs treated for B canis and evaluate the performance of the quantitative serologic canine Brucella multiplex (CBM) assay for monitoring treatment response., Animals: Diagnostic records from the Animal Health Diagnostic Center at Cornell University were retrospectively reviewed (2017-2022) for dogs that underwent repeat B canis serologic testing. Medical records were requested to compare the clinical presentations and outcomes for dogs that underwent treatment for B canis. Changes in CBM antibody values were compared between dogs with and without resolution of clinical signs., Results: While treatment protocols varied in the 30 treated dogs meeting the inclusion criteria, poly-antimicrobial therapy was prescribed in 97% (29/30) of cases. Gait abnormalities, spinal pain, and discospondylitis were the most common clinical abnormalities. A difference (P value = .0075) in the percent decrease in CBM assay PO1 antibody values was found in dogs with resolved clinical signs., Clinical Relevance: Young dogs presenting with recurring lameness or back pain should be screened for B canis infection. A 40% decline in CBM assay values 2 to 6 months posttreatment can be supportive of response to treatment. Further prospective studies are needed to determine the ideal B canis treatment regimen and the magnitude of public health risks associated with maintaining neutered B canis-infected animals as pets.

Brucellosis is a highly infectious zoonotic disease of global significance due to its adverse impact on public health, economics, and trade. Despite being one of the most prevalent zoonoses worldwide, attention given to global brucellosis control and prevention has been inadequate. Brucella species of greatest one-health relevance in the US include those infecting dogs (Brucella canis), swine (Brucella suis), and cattle and domestic bison (Brucella abortus). Although not endemic in the US, Brucella melitensis warrants awareness as it poses a risk to international travelers. While brucellosis has been eradicated from domestic livestock in the US, its detection in US companion animals (B canis) and US wildlife reservoirs (B suis and B abortus) and enzootic presence internationally pose a threat to human and animal health, warranting its spotlight on the one-health stage. The challenges of B canis diagnosis in humans and dogs is addressed in more detail in the companion Currents in One Health by Guarino et al, AJVR, April 2023. Human consumption of unpasteurized dairy products and occupational exposure of laboratory diagnosticians, veterinarians, and animal care providers are responsible for human exposures reported to the US CDC. Diagnosis and treatment of brucellosis is challenging due to the limitations of diagnostic assays and the tendency of Brucella spp to produce nonspecific, insidious clinical signs and evade antimicrobial therapy, making prevention essential. This review will focus on zoonotic considerations for Brucella spp found within the US along with their epidemiology, pathophysiology, clinical presentation, treatment, and control strategies.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four Brucella strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: Brucella abortus ( B. abortus ), B. melitensis, B. suis (smooth strains) and B. canis (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of B. canis antibodies. The aim of our study was to identify smooth Brucella strain antibodies in canines. We hypothesize that the Brucella abortus Fluorescence Polarization Assay would be successful in identifying smooth Brucella strain antibodies in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Brucella Slide Agglutination Test (CBSA), Brucella canis Agar Gel Immunodiffusion II test (AGIDII), Brucella abortus Card Agglutination Test (BCA), and the Brucella abortus Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth Brucella strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough Brucella strain serology results (0-2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of Brucella by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018 to 2019 via Brucella abortus FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant ( p < 0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs, respectively., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Helms, Balogh, Franklin-Guild, Lahmers, Caswell and Cecere.)

Whole-genome sequencing of Mycoplasma mucosicanis type strain 1642 was performed to support efforts to better understand the clinical significance of Mycoplasma infection in canine health. The availability of this sequence will also further the development of highly specific diagnostic tests., (Copyright © 2019 Tallmadge et al.)