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Monofluoroacetate is a highly lethal toxin that causes death by inhibiting cellular adenosine triphosphate (ATP) production. The heart and brain are the primary target organs. Acute death is attributed to cardiac fibrillation and/or convulsions. Although it occurs naturally in some plants, a major source of animal intoxication is access to sodium monofluoroacetate (NaMFA) pesticide, which continues to be a concern in the USA and around the world despite restricted use in some countries including the USA. There are also concerns about misuse of this pesticide for malicious poisoning. Currently, a tissue-based diagnostic method for NaMFA intoxication in animals is lacking. There is a critical need by the veterinary diagnostic community for a simple, sensitive and reliable tissue-based diagnostic test to confirm NaMFA poisoning in animals. We have developed and extensively evaluated a sensitive novel liquid chromatography combined with tandem mass spectrometry method suitable for this purpose. The limits of detection and limits of quantitation are 1.7 and 5.0 ng/g, respectively. The accuracy and precision met or exceeded expectations. The method performance was verified using the incurred kidney obtained from animal diagnostic cases. This novel kidney-based method is now available for clinical use and can help with diagnostic purposes, including detecting potential issues related to animal foods. [ABSTRACT FROM AUTHOR]

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates. [ABSTRACT FROM AUTHOR]

Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3–3.1 ng/g, and the limits of quantification were 0.8–9.4 ng/g. The recoveries obtained using UPLC-MS were 90–115%, and relative SDs were 1.2–13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86–118%, with relative repeatability SDs of 3.7–11%, relative reproducibility SDs of 7.8–31.2%, and Horwitz ratio values of 0.5–1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods. [ABSTRACT FROM AUTHOR]

Background: Throughout the COVID-19 pandemic, veterinary diagnostic laboratories have tested diagnostic samples for SARS-CoV-2 both in animals and over 6 million human samples. An evaluation of the performance of those laboratories is needed using blinded test samples to ensure that laboratories report reliable data to the public. This interlaboratory comparison exercise (ILC3) builds on 2 prior exercises to assess whether veterinary diagnostic laboratories can detect Delta and Omicron variants spiked in canine nasal matrix or viral transport medium. Methods: The ILC organizer was an independent laboratory that prepared inactivated Delta variant at levels of 25 to 1000 copies per 50 µL of nasal matrix for blinded analysis. Omicron variant at 1000 copies per 50 µL of transport medium was also included. Feline infectious peritonitis virus (FIPV) RNA was used as a confounder for specificity assessment. Fourteen test samples were prepared for each participant. Participants used their routine diagnostic procedures for RNA extraction and real-time reverse transcriptase-PCR. Results were analyzed according to International Organization for Standardization (ISO) 16140–2:2016. Results: Overall, laboratories demonstrated 93% detection for Delta and 97% for Omicron at 1000 copies per 50 µL. Specificity was 97% for blank samples and 100% for blank samples with FIPV. No differences in Cycle Threshold (Ct) values were significant for samples with the same virus levels between N1 and N2 markers, nor between the 2 variants. Conclusions: The results indicated that all ILC3 participants were able to detect both Delta and Omicron variants. The canine nasal matrix did not significantly affect SARS-CoV-2 detection. [ABSTRACT FROM AUTHOR]

Anti-coagulant rodenticides (ARs) are commonly utilized for controlling rodent populations; however, non-target companion and wildlife animals are also exposed. A method was developed for quantitation of seven ARs (chlorophacinone, coumachlor, bromadiolone, brodifacoum, difethialone, diphacinone and warfarin) and dicoumarol (a naturally occurring anti-coagulant) in animal serum. Analytes were extracted with 10% (v/v) acetone in methanol and analyzed by reverse phase high-performance liquid chromatography–tandem mass spectrometry using electrospray ionization (negative mode) combined with multiple reaction monitoring. In-house method validation in the originating laboratory using non-blinded samples revealed method limits of quantitation at 2.5 ng/mL for all analytes. The inter-assay accuracy ranged from 99% to 104%, and the relative standard deviation ranged from 3.5% to 20.5%. Method performance was then verified in the originating laboratory during an exercise organized by an independent party using blinded samples. The method was successfully transferred to two naïve laboratories and further evaluated for reproducibility among three laboratories by means of Horwitz ratio (HorRat(R)) values. Such extensive validation provides a high degree of confidence that the method is rugged, robust, and will perform as expected if used by others in the future. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans. [ABSTRACT FROM AUTHOR]

Aim: The aim of the study is to determine the basic conceptual, methodological and organizational-methodological aspects of the process of forming health competence of students using natural physiotherapy, to formalize them in the form of a preventive model. Materials and Methods: The pedagogical experiment lasted during 2019-2021 in Sumy State Pedagogical University named after A.S. Makarenko. The pedagogical research was attended by 128 first- and second-year students majoring in «Physical Education», «Physical Therapy, Occupational Therapy» Sumy State Pedagogical University named after A.S. Makarenko. The research methods were as follows: analysis of special and scientific literature, pedagogical observation, pedagogical experiment, pedagogical testing, methods of statistical data processing. Results: In order to organize and systematize this theoretical and practical experience, we have developed a preventive model of forming health-save competence of students, which allows to form an idea of the determinants of health, ways to strengthen it, guide students to follow the rules of healthy living and application of a wide range of natural physiotherapeutic means. Conclusions: The pedagogical model of formation of health-save competence orients students on observance of rules and norms of a healthy way of life with application of natural physiotherapeutic means (physical exercises, water procedures, air, solar heat and light), systematizes and formalizes representations of integral, multilevel, integrative, interdisciplinary educational process in the conditions of educational institution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 500 ng/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 HorRatr 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. [ABSTRACT FROM AUTHOR]

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 "RosaVR 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Aflatoxins (AFs) are secondary metabolites of fungi and are one of the causes of toxin-related pet food recalls. An intralaboratory method was previously developed to quantify aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in animal liver by HPLC with fluorescence detection. Objective: The aim of this study was to extensively evaluate the method performance with a single-laboratory blinded method test (BMT-S) and a multilaboratory blinded method test (BMT-M). Methods: Blinded tissue samples were prepared by a third-party laboratory and sent out to participating laboratories for both BMT-S and BMT-M. Results: In both tests, participants analyzed blinded samples prepared by an independent laboratory. In the BMT-S, accuracy ranged between 111 and 154% for AFB1 and 113 and 159% for AFM1 within the quantitation range of 0.1-0.5 ng/g. The HorRat values for repeatability ranged between 0.1 and 0.3 for AFB1 and 0.3 and 0.6 for AFM1. In the BMT-M, the interlaboratory accuracy ranged between 77 and 81% for AFB1 and 83 and 85% for AFM1 within the quantitation range of 0.2-10 ng/g. The HorRat values for reproducibility ranged between 0.4 and 0.7 for AFB1 and 0.4 and 0.9 for AFM1. Both recovery and reproducibility were acceptable. Conclusions: BMT-M evaluation demonstrated that the method was suitable for quantitation of aflatoxins B1 and M1 in animal liver between laboratories. Highlights: The BMT-S and BMT-M results demonstrated that the method is rugged and reproducible among the participating laboratories. [ABSTRACT FROM AUTHOR]

Abstract: Effects of the dosing matrix and timing on the onset of renal crystal formation were evaluated in male and non-pregnant female rats (Fisher 344) exposed to both melamine (MEL) and cyanuric acid (CYA) for 28days. Rats were fed ground feed containing 60ppm MEL and 60ppm CYA, (5mg/kgbw/day equivalent), or exposed via oral gavage to carboxymethylcellulose containing 5mg/kgbw MEL followed by 5mg/kgbw CYA either consecutively (<1min apart) or delayed 45min after MEL. Staggered gavage exposure to MEL/CYA caused extensive renal crystal formation as compared to when the two compounds were administered consecutively or in feed. Treatment related effects included reduced weight gain, feed consumption, and testicular weight and increased kidney weight, water consumption and urine output. Animals from the staggered MEL/CYA gavage exposure group became ill and were removed after 9days of exposure. Approximately 1week after the initiation of exposure microscopic urinalysis revealed MEL/CYA crystals in both groups of gavaged animals but not in the MEL/CYA feed treatment groups. Urinary crystals were smaller (10μm) in animals consecutively gavaged. In contrast the urinary crystals were larger (20–40μm) and frequently clumped in the animals in the staggered gavage group. [Copyright &y& Elsevier]

Background: Aflatoxins (AFs) are secondary metabolites of fungi and are one of the causes of toxin-related pet food recalls. An intralaboratory method was previously developed to quantify aflatoxin B 1 (AFB 1 ) and aflatoxin M 1 (AFM 1 ) in animal liver by HPLC with fluorescence detection. Objective: The aim of this study was to extensively evaluate the method performance with a single-laboratory blinded method test (BMT-S) and a multilaboratory blinded method test (BMT-M). Methods: Blinded tissue samples were prepared by a third-party laboratory and sent out to participating laboratories for both BMT-S and BMT-M. Results: In both tests, participants analyzed blinded samples prepared by an independent laboratory. In the BMT-S, accuracy ranged between 111 and 154% for AFB 1 and 113 and 159% for AFM 1 within the quantitation range of 0.1-0.5 ng/g. The HorRat values for repeatability ranged between 0.1 and 0.3 for AFB 1 and 0.3 and 0.6 for AFM 1 . In the BMT-M, the interlaboratory accuracy ranged between 77 and 81% for AFB 1 and 83 and 85% for AFM 1 within the quantitation range of 0.2-10 ng/g. The HorRat values for reproducibility ranged between 0.4 and 0.7 for AFB 1 and 0.4 and 0.9 for AFM 1 . Both recovery and reproducibility were acceptable. Conclusions: BMT-M evaluation demonstrated that the method was suitable for quantitation of aflatoxins B 1 and M 1 in animal liver between laboratories. Highlights: The BMT-S and BMT-M results demonstrated that the method is rugged and reproducible among the participating laboratories.