Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism

Abstract

Liver is a reliable matrix for laboratory confirmation of avian botulism using real-time PCR. Here, we developed, optimized, and validated the analytical steps preceding PCR to maximize the detection of Clostridium botulinum group III in avian liver. These pre-PCR steps included enrichment incubation of the whole liver (maximum 25 g) at 37°C for at least 24 h in an anaerobic chamber and DNA extraction using an enzymatic digestion step followed by a DNA purification step. Conditions of sample storage before analysis appear to have a strong effect on the detection of group III C. botulinum strains and our results recommend storage at temperatures below -18°C. Short-term storage at 5°C is possible for up to 24 h, but a decrease in sensitivity was observed at 48 h of storage at this temperature. Analysis of whole livers (maximum 25 g) is required and pooling samples before enrichment culturing must be avoided. Pooling is however possible before or after DNA extraction under certain conditions. Whole livers should be 10-fold diluted in enrichment medium and homogenized using a Pulsifier® blender (Microgen, Surrey, UK) instead of a conventional paddle blender. Spiked liver samples showed a limit of detection of 5 spores/g liver for types C and D and 250 spores/g for type E. Using the method developed here, the analysis of 268 samples from 73 suspected outbreaks showed 100% specificity and 95.35% sensitivity compared with other PCR-based methods considered as reference. The mosaic type C/D was the most common neurotoxin type found in examined samples, which included both wild and domestic birds.

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

“…Liver was the only matrix tested here because it is used for routine confirmation of avian botulism in our laboratory [15,24]. Using the conventional method, isolation was reported to be easier from liver than from caeca [27].…”

“…Besides the assay with the method depicted in Figure 3 using naturally contaminated samples, samples spiked with a known initial spore contamination level and of a known BoNT type were used to confirm that the method is indeed appropriate for isolating C. botulinum group III strains involved in avian botulism outbreaks. A method had been previously developed and validated for detecting C. botulinum in avian liver using PCR with a view of diagnosing avian botulism [24]. Its limit of detection was determined as five spores per gram of liver.…”

“…An overview of the outbreaks diagnosed by the French NRL for avian botulism was compiled to be able to select representative samples of situations encountered in the field in order to develop and evaluate the method. All outbreaks confirmed by the French NRL (clinical signs and a positive real-time PCR detection in samples collected from animals [24]) between 2013 and 2019 were listed and are presented in Figure 1.…”

“…According to a previously optimized method [24], livers (≤25 g) were ten-fold diluted in TPGY and incubated for at least 24 h at 37 • C in an anaerobic chamber (A35, Don Whitley distributed by Biomérieux, Bruz, France). DNA was extracted using 1 mL of this enrichment broth and BoNT genes were detected using real-time PCR.…”

About the Evaluation of the Effectiveness of Various Methods of Diagnosis, Prediction and Classification Presented in the Literature

“…Note that in the literature and on many sites, indeed, there are often unfounded judgments that sensitivity and specificity are the main parameters for evaluating the effectiveness of different methods, and accuracy, predictability, and others are secondary. Apparently, such an understanding of the issue in the literature is quite common, judging by the fact that in a large number of works, either one parameter or two are used to evaluate the effectiveness of the methods, and mostly sensitivity and specificity [13,14]. Further, it will be shown that these are fundamentally incorrect judgments, since operating with such canonical concepts as sensitivity and specificity does not lead to an unambiguous assessment of the effectiveness of the methods.…”