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.

Fig 1. Sample analysis workflow for suspected avian botulism cultures.

Parameters evaluated during the study: test sample type (whole organ means that the whole liver or up to 25g of the liver was analyzed), enrichment culturing conditions, DNA extraction methods. The CFX96 thermocycler (Bio-Rad, Marne-la-Coquette, France) was used for Real-Time PCR. Analyses performed by ANSES are indicated in black, and performed by LABOCEA are in grey. Kit 1: QIAamp^® DNA Mini kit (Qiagen, Courtaboeuf, France), Kit2: InstaGene Matrix (Bio-Rad, Marne-la-Coquette, France), Kit3: Mericon Bacteria+ (Qiagen, Courtaboeuf, France). CII, CIII, DII, DIII, E are the primers and probe used to perform real-time PCR for the detection of type C, D, C/D, D/C and E BoNT genes [4, 14]. L: Liver; L1 to L5: Liver N° 1 to Liver N° 5. M1 to M4: Method 1 to method 4.

Fig 2. Confirmation of avian botulism suspicions using different types of test samples.

Method 1: the entire liver was analyzed (max 25 g), Method 2: 1 g of each single liver was analyzed, Method 3: an aliquot of each liver was sampled after its homogenization in Bagfilter^® (Biomérieux, Craponne, France) and separated into two groups and 1 g of both pools were analyzed, Method 4: a slice of each liver was sampled and 1 g of this pool was analyzed. For each type of test sample, the number of suspected outbreaks detected is shown in black, the number of suspected outbreaks not detected is shown in gray, and the number of suspected outbreaks not tested is shown in white.

Fig 3. Percentage of detection of 5 or 50 spores of C. botulinum strain 109 785 (type C) or 105 256 (type D) in spiked liver samples after A) storage of spiked samples for 7 days at 20°C, 5°C or at a temperature below -18°C (n = 9 for each condition); B) storage of spiked samples at 5°C for 0 h, 24 h and 48 h (n = 6 for each condition), C) storage at a temperature below -18°C for 1 month and 6 months (n = 6 for each condition).

Fig 4. Detection of 5 or 50 spores per gram of liver of C. botulinum strain 109 785 (type C) or 105 256 (type D) in spiked liver samples after homogenization with either Pulsifier^® blender (Microgen, Surrey, UK) (in white) or paddle blender (in gray) (n = 9 for each condition) and after incubation for 24 h in an anaerobic chamber at 37°C.

A: detection of C. botulinum after homogenization with Pulsifier^® (Microgen, Surrey, UK) or paddle blender, B: boxplot of the threshold cycle number (Ct) obtained when livers were spiked with 50 spores, C: boxplot of Ct obtained when livers were spiked with 5 spores. CII, CIII, DII, DIII: names of the primers used for the detection of type C (CII, CIII) and type D (DII and DIII) C. botulinum. P: Pulsifier^® blender (Microgen, Surrey, UK), S: Paddle blender.

Fig 5. Percentage of detection of 5 or 50 spores of C. botulinum strain 109 785 (type C) or 105 256 (type D) in spiked liver samples after incubation for 24 h at 37°C in an anaerobic chamber (A35, Don whitley, distributed by Biomérieux, Bruz, France), in an anaerobic container with gas or with a Gas-pak (AnaeroGen, Oxoid, Dardilly, France) (n = 6 for each condition).

Fig 6. Percentage of detection of 5 or 50 spores of C. botulinum strain 109 785 (type C) or 105 256 (type D) in spiked liver samples after incubation for 24 h in an anaerobic container with gas at 30°C, 37°C and 41.5°C (n = 6 for each condition).

Fig 7. Diagnosis scheme for avian botulism by detection of C. botulinum in livers using real-time PCR.

Parameters optimized in this study are shown in bold.