Campylobacter jejuni is a leading cause of human foodborne illness, and pet dogs can be a source of infection. Despite recognition of this zoonotic potential, tests for detection of C. jejuni are not performed routinely in dogs. We compared bacterial culture and molecular methods for the detection of C. jejuni directly from canine feces and from Bolton enrichment broth inoculated with feces in a multi-laboratory randomized-method test. Although culture offers the advantage of collection of isolates for further epidemiologic study, real-time PCR (rtPCR) to detect cpn60 from DNA isolated directly from canine feces was more sensitive than culture and more accurate than rtPCR using DNA isolated from enrichment cultures. The limit of detection for our cpn60 rtPCR was 320 cfu/g.

A 2016-2018 outbreak in the United States demonstrated the importance of pet dogs as a source of Campylobacter jejuni. Canine feces are not tested routinely for C. jejuni. We previously compared culture-based and molecular methods for detection of C. jejuni and found that a cpn60 real-time PCR (rtPCR) assay using DNA isolated directly from canine feces was more reliable than culture, with a limit of detection (LOD) of 320 cfu/g. Later, during an outbreak investigation, a gyrA rtPCR assay had a LOD of 40 cfu/g. In our inter-laboratory comparison exercise, 27 analysts at 19 laboratories successfully detected C. jejuni in canine feces using the gyrA rtPCR. Samples inoculated with 45,600 cfu/g were correctly identified as positive by 100% of analysts. Samples inoculated with 4,700, 460, or 200 cfu/g were correctly identified by 96%, 77%, and 40% of analysts, respectively. The addition of C. jejuni gyrA rtPCR to previous methods allows laboratories to rapidly identify C. jejuni-positive canine fecal samples at a lower LOD. Isolation of DNA directly from feces rather than from enrichment broth offers the advantage of fewer opportunities for cross-contamination during testing and decreased time between specimen collection and test completion. The updated method, with the addition of the gyrA rtPCR, offers increased sensitivity and will ultimately improve overall detection of C. jejuni in canine feces at veterinary diagnostic laboratories, further supporting canine and public health.

The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a collaborative network established in 2010, is a partnership between the Food and Drug Administration’s Center for Veterinary Medicine (FDA CVM) and 48 veterinary diagnostic laboratories (VDLs) across North America. Vet-LIRN actively supports the CVM mission of protecting human and animal health by leveraging its network of VDLs. Initially focused on issues in animal foods, including by testing animal diagnostic samples, Vet-LIRN now addresses a broad range of CVM’s priorities. These include responding to animal foodborne illness outbreaks, developing new methods to detect potential microbial and chemical contaminants in animal foods, tracking antimicrobial resistance (AMR), promoting antimicrobial stewardship in veterinary medicine, and preparing for emerging One Health threats such as COVID-19 and Highly Pathogenic Avian Influenza (HPAI). Over the past 15 years, Vet-LIRN has played a pivotal role in many high-profile and important public health success stories, such as responding to multidrug-resistant Campylobacter outbreaks in puppies, aflatoxin contamination in pet food, Salmonella in pig ear treats, and botulinum toxin in alfalfa cubes. Additionally, Vet-LIRN’s AMR monitoring program collects data to understand AMR trends and assist in the response to foodborne and zoonotic outbreaks. Through collaboration with other key stakeholders such as CVM regulatory colleagues and external partners at the United States Department of Agriculture (USDA) and the Centers for Disease Control and Prevention (CDC), Vet-LIRN ensures rapid responses to critical issues. Looking ahead, Vet-LIRN remains dedicated to continuous improvements, reinforcing its commitment to the sustained protection of human and animal health.

The culture method was developed and validated by 11 participating Vet-LIRN laboratories as a collaborative effort under the grant Evaluation of Salmonella in Symptomatic and Asymptomatic Pets: Study for the Vet-LIRN Program (https://grants.nih.gov/grants/guide/rfa-files/rfa-fd-11-010.html). This method is based on ISO 6579:2002 Annex D- “Detection of Salmonella spp. in animal feces and in samples of the primary production stage” with some modifications

The PCR method was developed and validated by 11 participating Vet-LIRN laboratories as a collaborative effort under the grant Evaluation of Salmonella in Symptomatic and Asymptomatic Pets: Study for the Vet-LIRN Program (https://grants.nih.gov/grants/guide/rfa-files/rfa-fd-11-010.html).

Abstract Background Aflatoxins (AFs) are common feed contaminants and are one of the common causes of toxin-related pet food poisoning and recalls. Objective Currently, there are no validated methods for the detection and quantitation of AFs in biological matrices to diagnose AF exposure in live animals. Following a successful intra-laboratory method development to quantify AFB1 and AFM1 in animal urine by HPLC with fluorescence detection (HPLC–FLD), the present study was conducted to extensively evaluate the method performance in an unbiased manner using blinded samples. Methods The evaluation included two stages. First, the performance was verified in the method-originating laboratory in a single-laboratory blinded method test (BMT-S) trial followed by a multi-laboratory blinded method test (BMT-M) trial. Results In both trials, accuracy, repeatability, and reproducibility were satisfactory confirming the relatively good ruggedness and robustness of the method and ensuring that it will perform as expected if used by other laboratories in the future. Conclusions We extensively evaluated the performance of a quantitative method to detect AFB1 and AFM1 in animal urine by HPLC-FLD by two different laboratories in two separate BMT-S and BMT-M trials. Both BMT results demonstrated the satisfactory accuracy and precision of the method. It is now available to be adopted by other diagnostic laboratories for purposes of diagnosing AF intoxication in animals. Highlights A simple urine-based diagnostic test method using HPLC–FLD that originated in a single laboratory now has passed a multi-laboratory evaluation and is now available to be shared with other diagnostic laboratories for purposes of diagnosing AF intoxication in animals so better treatment can be rendered.

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.

Objective Carbapenems are broad-spectrum β-lactams with excellent activity against multidrug-resistant (MDR) Enterobacterales. Unfortunately, resistance to carbapenems within this bacterial family, known as carbapenem-resistant Enterobacterales (CRE), occurs and challenges the ability to treat difficult MDR infections. Although the impact of carbapenem-resistance has been greatest in human medicine, reports in the veterinary literature are increasing especially as national veterinary antimicrobial resistance surveillance programs are now in place. In this brief communication, we report the isolation of a non-carbapenemase-producing, carbapenem-resistant Klebsiella pneumoniae from the urine of a dog, discuss the likely mechanism of resistance, and wider implications. Animal Canine. Procedure Whole genome sequencing and phenotypic antimicrobial susceptibility testing was performed on a K. pneumoniae isolated from the urine of a dog. Results Antimicrobial susceptibility testing identified phenotypic resistance to imipenem and meropenem. Phenotypic detection of carbapenemase production was negative. Whole genome sequencing identified efflux pump genes associated with carbapenem resistance and point mutations in membrane porin genes. No carbapenemase gene was identified. Conclusion Phenotypic antimicrobial susceptibility testing identified the K. pneumoniae as a non-carbapenemase producing carbapenem-resistant organism with the proposed genotypic mechanism including alteration of efflux pumps and membrane porin activity and/or expression. Clinical significance Currently, there is limited use of carbapenem antimicrobial drugs in veterinary medicine, and practitioners may be unfamiliar or unaware of this type of resistance, its significance on routine antimicrobial susceptibility test reports, and implications for antimicrobial therapy and public health. Carbapenem-resistant Enterobacterales are infrequently isolated from companion animals; however, due to increasing adoption of advanced medical and surgical interventions, they may become more prevalent.

The U.S. Food and Drug Administration’s (FDA′s) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening.

Four previously healthy adult domestic shorthair cats (2 male, 2 female) from one household developed acute vomiting and ataxia less than 12 hours after consuming a commercial canned cat food. Blood work abnormalities included mild hyperglycemia with increased alanine aminotransferase (n = 1) and decreased blood urea nitrogen (n = 2). The veterinarian conducted whole blood ethylene glycol (EG) tests, which were positive for all cats. There were no known EG exposures. All cats were treated for suspected EG toxicosis and fully recovered after 48 hours. Separately from the cats' case, the same food was voluntarily recalled by the manufacturer 5 days later due to a higher-than-formulated amount of choline chloride added to the food. The 4 cats' canned cat food was tested for choline, choline chloride, EG, diethylene glycol, and propylene glycol to look for causes of the positive whole blood EG test. The cat food contained an average of 165,300 ppm (165,300 mg/kg) choline and 221,600 ppm (221,600 mg/kg) choline chloride on a dry matter basis, which is at least 65 times the recommended choline amount for adult cats. No glycols were detected. This case documents suspected choline toxicosis in cats after consuming a commercial canned cat food with a higher-than-formulated amount of choline chloride, and it suggests that choline toxicosis may cause a positive result on some EG whole blood tests. Choline toxicosis could be a possible differential diagnosis when a cat has a positive EG test and no known exposure to antifreeze.

BACKGROUND:Dogs are highly susceptible to aflatoxins, the mycotoxins which most commonly cause acute dog illnesses and deaths following the consumption of contaminated food. OBJECTIVE:In this study, a screening method to detect aflatoxin B1 (AFB1) in dry dog food was further evaluated at the FDA action level of 20 ng/g. A fourth-round multi-laboratory trial was performed. In contrast to the previous work, a different source of dog food was used in the multi-laboratory trial and more participants were involved. METHOD:The tested lateral flow method employs a modified procedure of the "Rosa® AFQ-Fast Test Kit" from Charm Sciences Inc. A total of 60 unfortified blank study samples, 220 study samples fortified at 20 ng/g, and 80 study samples fortified at 9-11 ng/g were prepared by an independent party and analyzed in 10 collaborating laboratories in a blinded manner. RESULTS:The pass rates were 98.3 and 94.5% for unfortified and 20 ng/g fortified study samples, respectively. CONCLUSIONS:The method is suitable for aflatoxin B1 screening at the FDA action level of 20 ng/g in a complex matrix such as dry dog food. HIGHLIGHTS:This work completes extensive method performance evaluation through four rounds of multi-laboratory trials.

Dietary exogenous thyrotoxicosis is infrequently observed in pet food. A retrospective evaluation of pet food investigations (PFI) was conducted for 17 dogs, including review of medical records, dietary and environmental exposure interviews, food testing, and regulatory action. Five PFIs occurring between 2016 and 2018 involved 7 food products including 2 food types, jerky treats or canned food, made from beef or bison. The dogs' serum thyroid hormone concentrations were evaluated before and after diet change. The foods were tested for active thyroid hormones and hormone precursors using high performance liquid chromatography with inductively coupled plasma mass spectrometry detection. The foods were also examined microscopically. Serum thyroid hormone concentrations of thyroxine (T4) varied depending on the food type consumed. Dogs that consumed dried jerky containing greater T4 concentrations often had increased serum T4 concentrations, whereas dogs that consumed canned products containing greater and 3,4,5- and 3,5,3'-triiodothyronine (T3) concentrations often had decreased serum T4 concentrations. After the diets were changed, serum T4 and T3 concentrations normalized at 1 month. Seven foods containing beef or bison had iodine concentrations greater than 11 mg/kg, and iodine speciation identified variable concentrations of iodide, T4, T3, monoiodotyrosine (MIT), and di-iodotyrosine (DIT). Thyroid gland was found in microscopic sections from one finished food and one ingredient, gullet. FDA performed Health Hazard Evaluations to categorize the exposure risk, and 5 foods were recalled for which the product packaging had not been discarded. Dietary exogenous thyrotoxicosis should be considered in dogs exhibiting clinical signs compatible with hyperthyroidism, especially if consuming beef-based food. A thyroid panel that includes serum iodine, coupled with a thorough feeding history can aid in diagnosis. Thyrotoxicosis is typically reversible after removing the contaminated food from the diet.

The Food and Drug Administration’s Veterinary Laboratory Investigation and Response Network is comprised of more than 40 animal diagnostic laboratories within North America and offers voluntary Proficiency Exercises to these participating laboratories. The joint Proficiency Exercise Program is run in collaboration with the Center for Food safety and Nutrition and Institute for Food safety and Health, located at the Moffett Proficiency Testing Laboratory. From 2012 to 2018, the Proficiency Exercise Program offered 20 proficiency tests or interlaboratory comparison exercises focused on veterinary analytes of interest. The program evaluated performance of laboratories, individual analysts, and the methods used. Over the six-year period, the program improved exercise schemes, as well as offered network laboratories exercises with analytes not routinely seen such as animal tissue with naturally occurring residues. Animal diagnostic laboratories can use performance results to assist with accreditation, demonstrate proficiency, and improve diagnostic capabilities.

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Background: Aflatoxins are one of the most heavily regulated mycotoxins in agriculture throughout the world. A variety of tests are used for detection, including rapid methods that are preferred when a large number of samples need to be quickly screened to implement an immediate action. However, a method developed for screening a specific commodity for the presence of mycotoxins requires further validation to demonstrate its suitability for additional matrices. Objective: In this study, a study was undertaken to evaluate a rapid screening method for aflatoxin B1 (AFB1) in dry dog food, a product potentially susceptible to aflatoxins contamination. Method: This test method employed lateral flow technology using kits obtained from Charm Sciences Inc. Three different sources of dry dog food were tested at the FDA action level of 20 ppb (ng/g) in three trials of a multi-laboratory study by four participants. A total of 80 unfortified blank samples, 270 samples spiked at 20 ppb, and 60 samples spiked below 20 ppb were analyzed. Results: The overall pass rates of 100% for unfortified samples and > 97% for 20 ppb-fortified samples meet the FDA guidance acceptance criteria for a limit test of 10-15% false positives and no more than 5% false negatives. Conclusions: The method is suitable for screening a large number of dry dog food samples for rapid response. Highlights: Multi-laboratory evaluation of a rapid method for aflatoxin screening in dog food.

Background: Ergot alkaloids are mycotoxins produced by the fungus Claviceps, which can contaminate grains and pose a health risk to humans and animals. Validation of an ergot alkaloid method in collaborative projects can be challenging due to instability of analytes, a lack of reliable reference materials, and a fully validated reference method. Objective: To extensively evaluate performance of a quantitative UHPLC-MS/MS method to detect ten ergot alkaloids at concentrations between 16 and 500ng/g in grains. Method: The method performance was evaluated in the Blinded Method Test (BMT) exercise, which allowed organizers to successfully address the challenges. Forty completely blinded test samples were prepared in an independent laboratory and shipped to a participating laboratory to analyze on two separate days. Results: Precision, accuracy, and HorRat(r) values met or exceeded the U.S. Food and Drug Administration recommendations. The design of the BMT exercise provided a high degree of confidence in data and conclusions drawn. Conclusions: The method performed in a manner as expected, and the method can be used by the laboratory for routine testing of wheat and rye grains. Highlights: BMT of laboratory methods facilitate validation of tests by evaluating performance in an unbiased manner.

We report isolation of a New Delhi metallo-β-lactamase-5–producing carbapenem-resistant Escherichia coli sequence type 167 from companion animals in the United States. Reports of carbapenem-resistant Enterobacteriaceae in companion animals are rare. We describe a unique cluster of blaNDM-5–producing E. coli in a veterinary hospital.

BackgroundAntimicrobial 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: 69Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60Staphylococcus pseudintermedius (dogs).ResultsWe 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%).ConclusionThe 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.

Dietary insufficiencies have been well documented to decrease growth rates and survival (and therefore overall production) in fish aquaculture. By contrast, the effects of dietary insufficiencies on the sensory biology of cultured fish remains largely unstudied. Diets based solely on plant protein sources could have advantages over fish-based diets because of the cost and ecological effects of the latter, but plant proteins lack the amino acid taurine. Adequate levels of taurine are, however, necessary for the development of a fully functional visual system in mammals. As part of ongoing studies to determine the suitability of plant-based diets, we investigated the effects of normal and reduced taurine dietary levels on retinal anatomy and function in European sea bass (Dicentrarchus labrax). We could not demonstrate any effects of dietary taurine level on retinal anatomy, nor the functional properties of luminous sensitivity and temporal resolution (measured as flicker fusion frequency). We did, however, find an effect on spectral sensitivity. The peak of spectral sensitivity of individuals fed a 5% taurine diet was rightward shifted (i.e., towards longer wavelengths) relative to that of fish fed a 0% or 1.5% taurine diet. This difference in in spectral sensitivity was due to a relatively lower level of middle wavelength pigment (maximum absorbance .500 nm) in fish fed a 5% taurine diet. Changes in spectral sensitivity resulting from diets containing different taurine levels are unlikely to be detrimental to fish destined for market, but could be in fishes that are being reared for stock enhancement programs.