ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

Sequence analysis was performed of all or part of the genes encoding the fusion (F), polymerase (L) and attachment (G) proteins of two French non-A/non-B avian pneumovirus (APV) isolates (Fr/85/1 and Fr/85/2). The two isolates shared at least 99.7% nt and 99.0% aa sequence identity. Comparison with the F genes from subgroup A, subgroup B or Colorado APVs revealed nt and aa identities of 70.0-80. 5% and 77.6-97.2%, respectively, with the L gene sharing 76.1% nt and 85.3% aa identity with that of a subgroup A isolate. The Fr/85/1 and Fr/85/2 G genes comprised 1185 nt, encoding a protein of 389 aa. Common features with subgroup A and subgroup B G proteins included an amino-terminal membrane anchor, a high serine and threonine content, conservation of cysteine residues and a single extracellular region of highly conserved sequence proposed to be the functional domain involved in virus attachment to cellular receptors. However, the Fr/85/1 and Fr/85/2 G sequences shared at best 56.6% nt and 31.2% aa identity with subgroup A and B APVs, whereas these isolates share 38% aa identity. Phylogenetic analysis of the F, G and L genes of pneumoviruses suggested that isolates Fr/85/1 and Fr/85/2 belong to a previously unrecognized APV subgroup, tentatively named D. G-based oligonucleotide primers were defined for the specific molecular identification of subgroup D. These are the first G protein sequences of non-A/non-B APVs to be determined.

Fifty-six reverse transcriptions followed by a polymerase chain reaction (RT-PCR) were developed and/or assessed to detect and to type turkey rhinotracheitis virus (TRTV). Twenty-seven primers corresponding to sequences either common to both A and B viruses, or type-specific were respectively defined in the fusion (F), attachment (G) and nucleocapsid (N) proteins genes. Only one N-based RT-PCR detected 21/21 TRTVs isolated in four countries since 1985. Molecular typing (RT-PCR) and antigenic typing (ELISA) showed that TRTV strains antigenically related either to the 3BOC18 (UK/85/1) or to the 86004 (Fr/86/1) viruses belonged to the A or B genomic type respectively. Neither typing approach allowed assignment of two 1985 French isolates (Fr/85/1 and Fr/85/2) to either type A or B, these strains might thus belong to a third type. RT-PCR assays on tracheal and nasal swabs sampled during experimental and field infections significantly outperformed concurrent virus isolation in tissue culture and ELISA: G- and N-based RT-PCRs detected more positive samples than conventional methods. Molecular and serological results were concordant and demonstrated that all the recent French field viruses belonged to type B. Thus, N- and G-based RT-PCR are respectively specific and sensitive tools for rapid diagnosis and typing of TRTV in field samples.

Between 2011 and 2013, 17 poultry botulism outbreaks were investigated in France. All cases were associated with Clostridium botulinum type C-D. Presence of C. botulinum was studied in seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoirs, water, and feed. Swabs, litter, soil, darkling beetles, rodents and wild bird droppings, feed and water samples were collected. The presence of C. botulinum type C-D in the environment of affected flocks was detected in 39.5% of the 185 samples analysed by real-time polymerase chain reaction. C. botulinum type C-D was reported in each area. Four areas were more frequently contaminated, being found positive in more than one-half of farms: darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). After cleaning and disinfection, the ventilation system and/or the soil (in the houses and the surroundings) returned positive results in four out of eight poultry farms. Consequently, darkling beetles, the drinking water, the ventilation system and the soil in the surroundings and the houses were identified as the main critical contaminated areas to consider in poultry farms to prevent recurrence of botulism outbreaks.

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Animal botulism is caused by group III Clostridium botulinum strains producing type C and D toxins, or their chimeric forms C/D and D/C. Animal botulism is considered an emerging disease in Europe, notably in poultry production. Before our study, 14 genomes from different countries were available in the public database, but none were from France. In order to investigate the genetic relationship of French strains with different geographical areas and find new potential typing targets, 17 strains of C. botulinum group III were sequenced (16 from France and one from New Caledonia). Fourteen were type C/D strains isolated from chickens, ducks, guinea fowl and turkeys and three were type D/C strains isolated from cattle. The New Caledonian strain was a type D/C strain. Whole genome sequence analysis showed the French strains to be closely related to European strains from C. botulinum group III lineages Ia and Ib. The investigation of CRISPR sequences as genetic targets for differentiating strains in group III proved to be irrelevant for type C/D due to a deficient CRISPR/Cas mechanism, but not for type D/C. Conversely, the extrachromosomal elements of type C/D strains could be used to generate a genetic ID card. The highest level of discrimination was achieved with SNP core phylogeny, which allowed differentiation up to strain level and provide the most relevant information for genetic epidemiology studies and discrimination.

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.

A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.