EZ-SPOTs: A simple and robust high-throughput liquid handling platform.

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.

Human exposures to Brucella canis from a pregnant dog during an international flight: Public health risks, diagnostic challenges and future considerations.

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.

Serotype and anti-microbial resistance trends among bovine Salmonella isolates from samples submitted to a veterinary diagnostic laboratory in central New York, 2007-2021.

AIMS: Salmonella enterica is a leading cause of acute enteritis in people, and dairy cattle are an important reservoir of this pathogen. The objective of this study was to analyse serotype and anti-microbial resistance trends of Salmonella isolated from dairy cattle in the United States between 2007 and 2021. METHODS AND RESULTS: We collected data for bovine Salmonella isolates obtained from samples submitted to Cornell University's Animal Health Diagnostic Center (AHDC). We analysed 5114 isolates for serotype trends, and a subset of 2521 isolates tested for anti-microbial susceptibility were analysed for resistance trends. The most frequently identified serotypes were Salmonella Cerro, Dublin, Typhimurium, Montevideo, 4,[5],12:i:-, and Newport. Among these serotypes, a Cochran-Armitage trend test determined there was a significant increase in the proportion of isolates serotyped as Salmonella Dublin (p < 0.0001) and Montevideo (p < 0.0001) over time. There was a significant decrease in the proportion of isolates serotyped as Salmonella Cerro (p < 0.0001), Typhimurium (p < 0.0001), and Newport (p < 0.0001). For the anti-microbial resistance (AMR) analysis, we found an overall increase in the proportion of multi-drug-resistant isolates over time (p = 0.009). There was a significant increase in the proportion of isolates resistant to ampicillin (p = 0.007), florfenicol (p = 0.0002), and ceftiofur (p < 0.0001) and a marginal increase in resistance to enrofloxacin (p = 0.05). There was a significant decrease in the proportion of isolates resistant to spectinomycin (p = 0.0002), trimethoprim/sulphamethoxazole (p = 0.01), sulphadimethoxine (p = 0.003), neomycin (p < 0.0001), and gentamicin (p = 0.0002). CONCLUSIONS: Our results provide evidence of an increase in resistance to key anti-microbial agents, although the observed trends were driven by the sharp increase in the proportion of Salmonella Dublin isolates over time.

Assessing the urinary concentration of nitrofurantoin and its antibacterial activity against Escherichia coli, Staphylococcus pseudintermedius, and Enterococcus faecium isolated from dogs with urinary tract infections.

Nitrofurantoin, a broad-spectrum nitrofuran class antibiotic, is applied as a first-line antibiotic in treating human urinary tract infections (UTIs) due to its great efficacy and high achievable concentration. The interest in using this antibiotic in companion animals has increased due to the growing demand for effective antibiotics to treat UTIs caused by multidrug-resistant bacteria. Currently, the susceptibility interpretations for nitrofurantoin are based on the breakpoints set for humans, while the canine-specific breakpoints are still unavailable. In this study, we assessed the concentration of nitrofurantoin reaching the dog's urine using the recommended oral dosing regimen. In addition, we examined the efficacy of this breakpoint concentration against the common canine UTI pathogens, Escherichia coli, Staphylococcus pseudintermedius, and Enterococcus faecium. Eight experimental beagle dogs were treated with ~5 mg/kg of nitrofurantoin macrocrystal PO 8qh for 7 days. The urine samples were collected via cystocentesis at 2, 4, and 6 h after administration on day 2 and day 7 and used to quantify nitrofurantoin concentrations by ultra-high performance liquid chromatography. The results showed that 26.13-315.87 µg/mL nitrofurantoin was detected in the dogs' urine with a mean and median concentration of 104.82 and 92.75 µg/mL, respectively. Additionally, individual dogs presented with urinary nitrofurantoin concentrations greater than 64 µg/mL for at least 50% of the dosing intervals. This concentration efficiently killed E. coli, and S. pseudintermedius, but not E. faecium strains carrying an MIC90 value equal to 16, 16, and 128 µg/mL, respectively. Taken together, these results suggest that the value of 64 µg/mL may be set as a breakpoint against UTI pathogens, and nitrofurantoin could be an effective therapeutic drug against E. coli and S. pseudintermedius for canine UTIs.

A one-health review on brucellosis in the United States.

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.

Antibody response over time correlated with treatment outcome in 30 dogs naturally infected with Brucella canis (2017-2022).

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.

Effect of antimicrobial administration on fecal microbiota of critically ill dogs: dynamics of antimicrobial resistance over time.

BACKGROUND: Multidrug resistance in companion animals poses significant risks to animal and human health. Prolonged antimicrobial drug (AMD) treatment in animals is a potential source of selection pressure for antimicrobial resistance (AMR) including in the gastrointestinal microbiota. We performed a prospective study of dogs treated for septic peritonitis, pyometra, or bacterial pneumonia and collected repeated fecal samples over 60 days. Bacterial cultures and direct molecular analyses of fecal samples were performed including targeted resistance gene profiling. RESULTS: Resistant Escherichia coli increased after 1 week of treatment (D1:21.4% vs. D7:67.9% P < 0.001) and returned to baseline proportions by D60 (D7:67.9% vs D60:42.9%, P = 0.04). Dogs with septic peritonitis were hospitalized significantly longer than those with pneumonia or pyometra. Based on genetic analysis, Simpson's diversity index significantly decreased after 1 week of treatment (D1 to D7, P = 0.008), followed by a gradual increase to day 60 (D1 and D60, P = 0.4). Detection of CTX-M was associated with phenotypic resistance to third-generation cephalosporins in E. coli (OR 12.1, 3.3-68.0, P < 0.001). Lincosamide and macrolide-resistance genes were more frequently recovered on days 14 and 28 compared to day 1 (P = 0.002 and P = 0.004 respectively). CONCLUSION: AMR was associated with prescribed drugs but also developed against AMDs not administered during the study. Companion animals may be reservoirs of zoonotic multidrug resistant pathogens, suggesting that veterinary AMD stewardship and surveillance efforts should be prioritized.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

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.

Presumptive Identification of Smooth Brucella Strain Antibodies in Canines.

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.

Feline bacterial keratitis: Clinical features, bacterial isolates, and in vitro antimicrobial susceptibility patterns.

OBJECTIVE: To describe clinical characteristics, bacterial isolates, and in vitro antimicrobial susceptibility patterns for cats with bacterial keratitis. ANIMALS STUDIED: Eighty one cats with bacterial keratitis. PROCEDURES: Medical records of cats with a clinical diagnosis of bacterial keratitis, confirmed by corneal culture, were reviewed from June 2004 to July 2017. Animal signalment, bacterial isolates, aerobic bacteria in vitro antimicrobial susceptibility test results, and pertinent clinical features were recorded. Percentages of susceptible aerobic bacterial isolates were statistically compared among selected antimicrobials. RESULTS: There were 102 aerobic bacterial isolates recovered from corneal samples of the 81 cats presented with bacterial keratitis. The most frequent bacteria isolated were Staphylococcus species, which constituted 55% (56/102) of total isolates. All aerobic bacterial isolates grouped together had the highest percentage of susceptibility to ofloxacin (100%), ciprofloxacin (94%), chloramphenicol (93%), doxycycline (92%), ticarcillin (90%), gentamicin (89%), moxifloxacin (89%), tobramycin (86%), neomycin (85%), amikacin (84%), and cefazolin (84%). The same isolates had the lowest percentage of susceptibility to polymyxin B (2%), bacitracin (15%), and clindamycin (31%). When analyzed separately, the isolated Staphylococcus species had the highest percentage of susceptibility to ofloxacin (100%), tobramycin (93%), and neomycin (85%) and the lowest percentage of susceptibility to polymyxin B (3%) and bacitracin (13%). CONCLUSIONS: Members of the Staphylococcus genus were the most frequent bacteria isolated from cases of feline bacterial keratitis. On the basis of in vitro susceptibility testing and mechanism of action, ofloxacin, ciprofloxacin, ticarcillin, gentamicin, or moxifloxacin are recommended for initial antimicrobial therapy of suspected bacterial keratitis in cats.

Whole-Genome Sequence of the Mycoplasma mucosicanis Type Strain.

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.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Capnocytophaga keratitis in dogs: clinical, histopathologic, and microbiologic features of seven cases.

OBJECTIVE: To describe the clinical, microbiologic, and histopathologic features of Capnocytophaga keratitis in dogs. ANIMALS STUDIED: Seven dogs with naturally acquired Capnocytophaga keratitis. PROCEDURES: Medical records of dogs with a clinical diagnosis of keratitis and corneal cultures positive for Capnocytophaga spp. were reviewed. Dog signalment, medical history, clinical findings, and diagnostic assay results were recorded. RESULTS: Breeds included Boston terrier (n = 3 dogs), Rat terrier (n = 2), and single cases of mixed breed and Pug. All dogs examined had expansive corneal ulceration involving the majority of the corneal surface. Marked corneal infiltrates, keratomalacia, and hypopyon were present. Progression of corneal disease was rapid with extensive dissolution of the corneal stroma. Corneal lesions progressed to catastrophic perforations within 24 h of the initial examination in three dogs, requiring enucleation. One globe was enucleated after failure to resolve with long-term medical therapy. Globes and vision were retained in three dogs following aggressive medical therapy (two dogs) or 360° conjunctival graft surgery (one dog). Capnocytophaga cynodegmi, Capnocytophaga canimorsus, or unspeciated Capnocytophaga spp. were cultured from corneal samples of all dogs. Long, thin, gram-negative rods were present during cytological evaluation of the cornea in some dogs. Histopathologic evaluation of enucleated globes revealed severe and diffuse neutrophilic and collagenolytic keratitis. CONCLUSIONS: Capnocytophaga keratitis is a severe, rapidly progressive corneal infection in dogs that is associated with diffuse corneal involvement, extensive keratomalacia, and a relatively poor prognosis. Clinical features of canine Capnocytophaga keratitis are similar to human cases of this infection.

Detection of Salmonella spp. in veterinary samples by combining selective enrichment and real-time PCR.

Rapid screening for enteric bacterial pathogens in clinical environments is essential for biosecurity. Salmonella found in veterinary hospitals, particularly Salmonella enterica serovar Dublin, can pose unique challenges for culture and testing because of its poor growth. Multiple Salmonella serovars including Dublin are emerging threats to public health given increasing prevalence and antimicrobial resistance. We adapted an automated food testing method to veterinary samples and evaluated the performance of the method in a variety of matrices including environmental samples ( n = 81), tissues ( n = 52), feces ( n = 148), and feed ( n = 29). A commercial kit was chosen as the basis for this approach in view of extensive performance characterizations published by multiple independent organizations. A workflow was established for efficiently and accurately testing veterinary matrices and environmental samples by use of real-time PCR after selective enrichment in Rappaport-Vassiliadis soya (RVS) medium. Using this method, the detection limit for S. Dublin improved by 100-fold over subculture on selective agars (eosin-methylene blue, brilliant green, and xylose-lysine-deoxycholate). Overall, the procedure was effective in detecting Salmonella spp. and provided next-day results.

Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider.

A strain of lactic acid bacteria, designated 159469T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA-DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).