Biosecurity implementation on large-scale poultry farms in Europe: A qualitative interview study with farmers.

Biosecurity is an essential tool for rearing healthy animals. Biosecurity measures (BMs) are well known in poultry production, but it is difficult to assess actual implementation on farms. The aims of this qualitative study were (1) to provide an overview of biosecurity implementation according to poultry farmers in Europe; and (2) to better understand the reported reasons and potential obstacles for not implementing the measures. In seven European Union Member States, 192 farmers (118 under contract with a company and 68 independents) working in seven different categories of poultry production were interviewed on 62 BMs to determine the frequency of implementation and the reasons for non-implementation. Most of the replies (n = 7791) concerning BM implementation were reported by the farmers as "always" implemented (81%), statistically higher for breeders (87%) and layers (82%) and lower for independent farms versus farms under contract with a company (79.5% and 82.5%, respectively). Regardless the poultry production category, the most frequently implemented BMs declared by the farmers were daily surveillance of birds, rodent control and feed storage protection. Standard hygiene practices were also mentioned as high-implementation measures for most production categories, with some deficiencies, such as rendering tank disinfection after each collection and, for meat poultry, disinfection of the feed silo and bacterial control of house cleaning and disinfection between each cycle. The entry of vehicles and individuals onto poultry farms, especially during critical points of eggs collection for breeders and layers, as well as the presence of other animals, such as the "all in/all out" practice, particularly in layers and ducks, were also reported as the least commonly practiced measures. The main reasons for not implementing the measures (n = 1683 replies) were low awareness and poor knowledge of the expected benefits of biosecurity ("no known advantages" 14%, and "not useful" 12%), the lack of training ("not enough training" 5% and "advice" 7%), lack of time (19%), and financial aspects (17%). Despite the good overall biosecurity mentioned by the farmers, these findings highlight certain deficiencies, suggesting room for improvement and the need for targeted and tailored support of poultry farmers in Europe.

New Patterns for Highly Pathogenic Avian Influenza and Adjustment of Prevention, Control and Surveillance Strategies: The Example of France.

From 2020 up to summer 2023, there was a substantial change in the situation concerning the high pathogenic avian influenza (HPAI) virus in Europe. This change concerned mainly virus circulation within wildlife, both in wild birds and wild mammals. It involved the seasonality of HPAI detections, the species affected, excess mortality events, and the apparent increased level of contamination in wild birds. The knock-on effect concerned new impacts and challenges for the poultry sector, which is affected by repeated annual waves of HPAI arriving with wild migratory birds and by risks due to viral circulation within resident wild birds across the year. Indeed, exceeding expectations, new poultry sectors and production areas have been affected during the recent HPAI seasons in France. The HPAI virus strains involved also generate considerable concern about human health because of enhanced risks of species barrier crossing. In this article, we present these changes in detail, along with the required adjustment of prevention, control, and surveillance strategies, focusing specifically on the situation in France.

Human and animal botulism surveillance in France from 2008 to 2019.

Botulism is a human and animal neurological disease caused by the action of bacterial neurotoxins (botulinum toxins) produced by bacteria from the genus Clostridium. This disease induces flaccid paralysis that can result in respiratory paralysis and heart failure. Due to its serious potential impact on public health, botulism is a closely monitored notifiable disease in France through a case-based passive surveillance system. In humans, this disease is rare, with an average of 10 outbreaks reported each year, mainly due to the consumption of contaminated foods. Type B and to a lesser extend type A are responsible for the majority of cases of foodborne botulism. Each year, an average of 30 outbreaks are recorded on poultry farms, about 20 cases in wild birds and about 10 outbreaks in cattle, involving a large number of animals. Mosaic forms C/D and D/C in birds and cattle, respectively, are the predominant types in animals in France. Types C and D have also been observed to a lesser extent in animals. With the exception of botulinum toxin E, which was exceptionally detected throughout the period in wild birds, the types of botulism found in animal outbreaks are different from those identified in human outbreaks over the last ten years in France and no human botulism outbreaks investigated have been linked to animal botulism. In line with the One Health concept, we present the first integrative approach to the routine surveillance of botulism in humans and animals in France.

Two major epidemics of highly pathogenic avian influenza virus H5N8 and H5N1 in domestic poultry in France, 2020-2022.

The spread of highly pathogenic avian influenza (HPAI) viruses worldwide has serious consequences for animal health and a major economic impact on the poultry production sector. Since 2014, Europe has been severely hit by several HPAI epidemics, with France being the most affected country. Most recently, France was again affected by two devastating HPAI epidemics in 2020-21 and 2021-22. We conducted a descriptive analysis of the 2020-21 and 2021-22 epidemics, as a first step towards identifying the poultry sector's remaining vulnerabilities regarding HPAI viruses in France. We examined the spatio-temporal distribution of outbreaks that occurred in France in 2020-21 and 2021-22, and we assessed the outbreaks' spatial distribution in relation to the 2016-17 epidemic and to the two 'high-risk zones' recently incorporated into French legislation to strengthen HPAI prevention and control. There were 468 reported outbreaks during the 2020-21 epidemic and 1375 outbreaks during the 2021-22 epidemic. In both epidemics, the outbreaks' distribution matched extremely well that of 2016-17, and most outbreaks (80.6% and 68.4%) were located in the two high-risk zones. The southwestern high-risk zone was affected in both epidemics, while the western high-risk zone was affected for the first time in 2021-22, explaining the extremely high number of outbreaks reported. As soon as the virus reached the high-risk zones, it started to spread between farms at very high rates, with each infected farm infecting between two and three other farms at the peaks of transmission. We showed that the spatial distribution model used to create the two high-risk zones was able to predict the location of outbreaks for the 2020-21 and 2021-22 epidemics. These zones were characterized by high poultry farm densities; future efforts should, therefore, focus on reducing the density of susceptible poultry in highly dense areas.

Concomitant NA and NS deletion on avian Influenza H3N1 virus associated with hen mortality in France in 2019.

An H3N1 avian influenza virus was detected in a laying hens farm in May 2019 which had experienced 25% mortality in Northern France. The complete sequencing of this virus showed that all segment sequences belonged to the Eurasian lineage and were phylogenetically very close to many of the Belgian H3N1 viruses detected in 2019. The French virus presented two genetic particularities with NA and NS deletions that could be related to virus adaptation from wild to domestic birds and could increase virulence, respectively. Molecular data of H3N1 viruses suggest that these two deletions occurred at two different times.

Increasing incidence of Enterococcus-associated diseases in poultry in France over the past 15 years.

Enterococci are commensal intestinal bacteria and opportunistic pathogens in humans and animals. Enterococcus-associated diseases are an emerging health issue in poultry. The aim of this retrospective study was to assess the occurrence of enterococci in poultry in France with regard to the manifested diseases and the affected avian species. Our analysis is based on veterinary laboratory data collected by the French poultry epidemiological surveillance network (RNOEA) that monitors avian diseases in France based on the voluntary participation of its veterinarian members. Since the creation of the network in 1989, 12, 177 Enterococcus cases have been reported by veterinary laboratories (Enterococcus cecorum 53.1% and Enterococcus faecalis 24.3%), with emergence starting in 2006, year in which Enterococcus represented 0.4% of all reported pathogens, and incidence growing to 12.9% in 2020. The main diseases associated with these reports were locomotor disorders 35.2% (mainly involving E. cecorum 77.9%), septicaemia 34.9% (involving E. cecorum 53.4% and E. faecalis 23.8%), and omphalitis 14.4% (mainly involving E. faecalis 59.5%). Most of these Enterococcus-associated diseases (71.5%) were reported in broilers (particularly affected by the locomotor disorders and septicaemia involving E. cecorum), accounting for 9.1% of all the diseases reported in this production sector, with an increase from 1.4% in 2006 to 17.2% in 2020. This study highlights the emergence of enterococcal diseases in poultry in France over the past 15 years and the need to maintain a surveillance system.

Asymptomatic Carriage of C. botulinum Type D/C in Broiler Flocks as the Source of Contamination of a Massive Botulism Outbreak on a Dairy Cattle Farm.

In winter 2018, a massive type D/C cattle botulism outbreak occurred on a mixed dairy and broiler farm in France. An investigation was conducted based on the hypothesis of asymptomatic carriage in poultry. We set out to identify the source of contamination of the dairy cattle and to monitor the contamination of broilers over time, including the hatchery delivering chicks to the farm. Environmental samples were collected on the farm during the cattle outbreak (n = 40), after the outbreak for three successive broiler flocks (n = 128), and once in the hatchery delivering the chicks (n = 58). These samples were analyzed using real-time PCR after an enrichment step to detect Clostridium botulinum type D/C. The results showed contamination in the manure from the broilers raised just before the onset of the cattle outbreak (5 + /5), as well as in some of the components of the cattle ration (3 + /17). This latter contamination is likely due to the use of the same tractor bucket to remove litter from the poultry house and to prepare the cattle ration on the same day. Contamination monitoring over several months revealed continuous asymptomatic carriage in the broilers (4 + /20 and 17 + /20 cloacal swabs in 2 successive flocks), a persistence of C. botulinum type D/C in the ventilation system of the poultry house (8 + /14), and contamination of the equipment coming from the hatchery used for delivering the chicks (3 + /18). Further investigations conducted in the hatchery demonstrated contamination in the hatchery by C. botulinum type D/C (6 + /58). Comparison of samples using a multilocus variable number tandem repeat analysis showed the same profile for samples collected on broilers, cattle and in the hatchery. This study highlighted the crucial role of the implementation of biosecurity measures in mixed farms to avoid cross-contamination between production units given the potential asymptomatic carriage of poultry. This study also revealed the contamination of the poultry hatchery. Further investigations are required to better understand the role of hatcheries in the epidemiology of animal botulism.

Agreement between the categorization of isolates of Aeromonas salmonicida and Yersinia ruckeri by disc diffusion and MIC tests performed at 22℃.

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty-one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22 ± 2℃ and for the 177 Yersinia ruckeri they were read after 24-28 hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44-48 hr. Applying epidemiological cut-off values to the susceptibility data generated in these tests, the isolates were categorized as wild-type or non-wild-type. The agent-specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation.

Highly Pathogenic Avian Influenza A(H5N8) Virus Spread by Short- and Long-Range Transmission, France, 2016-17.

We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.

Epidemiological and Bacteriological Investigations Using Whole-Genome Sequencing in a Recurrent Outbreak of Pullorum Disease on a Quail Farm in France.

An outbreak of pullorum disease causing septicemia and high mortality was diagnosed in 2019 on a quail farm in western France. An initial episode had been detected in another building at the same site eight months earlier. Given the exceptional nature and the extent of the potential economic consequences of pullorum disease, epidemiological and bacteriological investigations using molecular sequencing tools were carried out. Salmonella Gallinarum and Salmonella Infantis were isolated (using the NF U 47-101 reference method) from samples taken from birds at the infected site. A resurgence of the initial episode by horizontal transmission of S. Gallinarum is the most likely hypothesis, supported by whole-genome sequencing (WGS) of the strains isolated during the two episodes. Risk health practices have been identified, including the rearing of animals of different ages and species on the same site. Recurrence is explained by the probable persistence of reservoirs of the pathogen on the site (manure, lesser mealworm beetles). The article also highlights the importance of decontamination measures, including pest control, as a key element in the success of the disease control protocol.

Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment.

Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV (A/goose/GD/1/1996, clade 2.3.4.4) were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization.IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment.

Manure contamination with Clostridium botulinum after avian botulism outbreaks: management and potential risk of dissemination.

BACKGROUND: Persistence of Clostridium botulinum in the environment is well known. Getting rid of it after animal botulism outbreaks is so tricky, especially as far as manure concerns. This study aimed at 1. describing manure management on 10 poultry farms affected by botulism and 2. assessing the persistence of C botulinum in poultry manure after the outbreak. METHODS: Each farm was visited twice at two different manure storage times (two weeks after manure removal and two months later). Fifteen samples of manure were collected on each visit and C botulinum was detected using real-time PCR. RESULTS: Management of manure varied among poultry farms (classical storage, addition of quicklime, bacterial flora or incineration). C botulinum was detected in the manure of all 10 farms, 56.5per cent of samples being positive. C botulinum was detected significantly more frequently at the second visit (65.8per cent vs 49.7per cent, P<0.01) and on the surface of the pile (63.1per cent vs 50per cent, P=0.025). CONCLUSION: This study shows the persistence of C botulinum in poultry manure over time after a botulism outbreak and highlights manure management as a key health issue in preventing spore dissemination in the environment and recurrence of the disease.

Cleaning and disinfection of crates and trucks used for duck transport: field observations during the H5N8 avian influenza outbreaks in France in 2017.

Transport of infected birds is thought to play a key role in the spread of avian influenza (AI) on poultry farms during epizootic outbreaks. Ensuring efficient cleaning and disinfection (C&D) of equipment used for transport is needed to prevent the spread of AI. This study aimed to evaluate the efficacy against the AI virus of C&D protocols applied on trucks and crates used for the transport of ducks during the H5N8 AI outbreaks in France in 2017. In 3 abattoirs, 16 transport vehicles and their crates were sampled by swabbing to detect the influenza type A genome by real-time reverse-transcription polymerase chain reaction. Vehicles were tested before and after decontamination, which was carried out in accordance with the abattoirs' protocols. A total of 86 samples out of 299 collected before C&D were positive for AI (29%); 7 trucks out of 16 transported crates detected positive for AI. After C&D, the AI genome was detected in 56 samples out of 308 (18%). Ten trucks were loaded with a shipment of AI-positive crates. Eight vehicles were detected positive in the cabin, on the truck bed, and/or on the wheels. Despite reinforcement of C&D, the efficacy of decontamination was variable among slaughterhouses. The efficacy seemed to depend on the initial contamination load, C&D protocols, and how the protocol is implemented. Breaks in biosecurity measures led to frequent contamination of trucks after C&D. Observational studies during animal health crises are of interest to analyze practices in emergency conditions and to put forward measures aimed at increased preparedness.

Husbandry Practices, Health, and Welfare Status of Organic Broilers in France.

Organic poultry production has increased sharply with growing consumer demand in the context of sustainable development. A study was conducted in 85 organic broiler flocks between 2014 and 2015 to describe the husbandry practices and the health and welfare status of organic broilers in France, and to study farming diversity by comparing independent farms (Ind farms, n = 15) with direct sales to farms working with companies (Comp farms, n = 70). Each flock was visited at 3 and 11 weeks of age to collect data on farming conditions, health disorders, and mortality. Welfare notation of 30 broilers per flock and parasitic examination of 5 broilers per flock was also performed. Findings showed significantly different farming management between Ind farms and Comp farms, with smaller flocks on the Ind farms (476 broilers/house vs. 3062 broilers/house, p < 0.01) more frequently in mobile houses. The mean mortality rate was 2.8%, mainly involving digestive disorders. Helminths were detected in 58.8% of the flocks. On average, 21.9% and 5.8% of broilers in a flock had footpad dermatitis and dirty feathers, respectively. The health and welfare characteristics of organic broilers on Ind farms vs. Comp farms were not significantly different, except dirtier feathers and more footpad dermatitis on Ind farms (19.1% vs. 2.9%, p = 0.03 and 39.6% vs. 18.1%, p = 0.02, respectively), associated with poultry housing conditions in mobile houses (p < 0.01). This study provides greater insight into farming sustainability aspects related to the husbandry practices, and the health and welfare of organic broilers in France.

Airborne Detection of H5N8 Highly Pathogenic Avian Influenza Virus Genome in Poultry Farms, France.

In southwestern France, during the winter of 2016-2017, the rapid spread of highly pathogenic avian influenza H5N8 outbreaks despite the implementation of routine control measures, raised the question about the potential role of airborne transmission in viral spread. As a first step to investigate the plausibility of that transmission, air samples were collected inside, outside and downwind from infected duck and chicken facilities. H5 avian influenza virus RNA was detected in all samples collected inside poultry houses, at external exhaust fans and at 5 m distance from poultry houses. For three of the five flocks studied, in the sample collected at 50-110 m distance, viral genomic RNA was detected. The measured viral air concentrations ranged between 4.3 and 6.4 log10 RNA copies per m3, and their geometric mean decreased from external exhaust fans to the downwind measurement point. These findings are in accordance with the possibility of airborne transmission and question the procedures for outbreak depopulation.

Dust exposure and health of workers in duck hatcheries.

OBJECTIVES: The objectives of this cross-sectional study were to investigate dust exposure and respiratory health of workers in duck hatcheries in western France. MATERIAL AND METHODS: Ninety volunteer workers, who work in sorting rooms and/or incubation rooms, participated in exposure assessments and medical examinations. Medical examinations were performed by occupational health practitioners.They filled-in a questionnaire with the workers, followed by a lung function test on each worker. General characteristics and prevalence of chronic respiratory symptoms were described in each type of working rooms. Associations between symptoms and exposure (working room or dust level) were studied in GEE multivariate models. RESULTS: Overall prevalence of chronic respiratory symptoms (cough, phlegm) and chronic bronchitis were similar or lower than in the reference population. However, prevalence of these symptoms was higher for those working in sorting rooms, that were associated with an increased risk of respiratory symptoms and decreased lung function. Respirable dust was also significantly associated with an increased risk of respiratory symptoms. The prevalence of asthma and rhinitis were well above those in the reference population, but did not vary among working rooms. Descriptive data suggested an occupational origin for some cases. CONCLUSIONS: Hatchery workers were at increased risk of compromised respiratory health due to dust exposure, particularly those who work in sorting rooms. Asthma and rhinitis were in excess in this population of workers. Thorough clinical examination of these workers should be performed and all exposures assessed.

Aeromonas Diversity and Antimicrobial Susceptibility in Freshwater-An Attempt to Set Generic Epidemiological Cut-Off Values.

The importance of the role of environment in the dissemination of antimicrobial resistant bacteria is now well recognized. Thus, bacterial indicators to monitor the phenomena are required. The Aeromonas genus is autochthonous in the aquatic environment and easy to detect in any water type, such as freshwater, or wastewater. These microorganisms are also causing infections in humans and animals (including fish). Furthermore, as Aeromonas spp. is able to acquire antimicrobial resistance mechanisms, it is candidate for indicator bacteria to follow antimicrobial resistance dissemination in aquatic environments. Unfortunately, to date, interpretation criteria for Aeromonas spp. for antimicrobial susceptibility tests are scarce in the literature. No epidemiological cut-off values for Aeromonas are currently available at EUCAST to interpret Minimum Inhibitory Concentrations (MIC). The only interpretation criteria available are clinical breakpoints from CLSI that are adapted from Enterobacteriaceae. Based on the results of MIC distributions obtained for a collection of environmental isolates of Aeromonas, this study aimed at proposing tentative epidemiological cut-off values (COWT) for Aeromonas spp. assessing whether the genus is an acceptable level of definition. Thus, 233 isolates collected from 16 rivers were identified at species level using Maldi-Tof (Bruker). Eleven different species were identified, the most abundant were A. bestiarum (n = 54), A. salmonicida (n = 45), A. sobria (n = 41), and A. eucrenophila (n = 37). 96-well micro-plates containing different concentrations of 15 antimicrobials, namely cefotaxime, ceftazidime, chloramphenicol, colistin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, nalidixic acid, oxolinic acid, streptomycin, temocillin, tetracycline, and trimethoprim-sulfamethoxazole, were prepared. The broth micro-dilution method was used to determine the antimicrobial susceptibility of each isolate. The estimation of COWT values was satisfactory obtained at genus level for all antimicrobials except cefotaxime and erythromycin. This first step is an invitation for other research teams to increase the amount of antimicrobial resistance data collected. Then, robustness of our proposed provisional generic epidemiological cut-off values could be assessed by testing antimicrobial susceptibility of various Aeromonas collections.

Enantiomer-specific accumulation and depuration of α-hexabromocyclododecane (α-HBCDD) in chicken (Gallus gallus) as a tool to identify contamination sources.

A LC-ESI(-)-HRMS method dedicated to the analysis of 6 HBCDD enantiomers at trace levels in animal matrices was developed, using a cellulose based stationary phase with a particle size of 2.5 µm. This method was applied to a sample set derived from a kinetic study of α-HBCDD previously conducted in fast- and slow-growing chickens (Gallus gallus domesticus, n = 49, plus controls), in order to study the enantiomer specific accumulation and depuration of α-HBCDD in various tissues. Regarding abdominal adipose tissue, muscle and liver, the average enantiomeric fractions of α-HBCDD (EFα) for continuously exposed groups ranged between 0.434 and 0.467, with standard deviations below 0.014, showing a significant enrichment in (-)α enantiomer even accentuated for slow growing individuals during depuration with EFα reduced by about 0.020. Similar trends were observed for pooled plasma. Then, EFα of circulating plasma α-HBCDD appeared to closely reflect EFα in storage tissues and liver, suggesting some equilibrium. The racemic elimination of α enantiomer in excreta during the contamination phase indicated that no preferential gastrointestinal absorption took place. By contrast, preferential excretion of (-)α-HBCDD from the circulating compartment to the intestinal lumen occurred during the depuration. Finally, the method was applied to samples collected in three chicken farms, selected for total HBCDD levels in muscle in the ng/g range, as a tool to determine whether the contamination occurred ante- or post-mortem, according to the chiral signature. Ante-mortem contamination was hypothesised for 2 farms, with feed being excluded as contamination source.

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

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.

Livers provide a reliable matrix for real-time PCR confirmation of avian botulism.

Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis.