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.

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.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.