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Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013 a
b
c
a
d
R. Souillard , C. Woudstra , C. Le Maréchal , M. Dia , M. H. Bayon-Auboyer , M. c
b
a
Chemaly , P. Fach & S. Le Bouquin a
Anses – UEB, Ploufragan-Plouzané Laboratory, Avian and Rabbit Epidemiology and Welfare Unit, Ploufragan, France b
Anses, Food Safety Laboratory, Maisons Alfort, France
c
Anses – UEB, Ploufragan-Plouzané Laboratory, Hygiene and Quality of Poultry and Pig Products Unit, Ploufragan, France d
Analysis and Development Laboratory 22 – LDA22/Labocea, Ploufragan, France Accepted author version posted online: 30 Aug 2014.Published online: 21 Oct 2014.
Click for updates To cite this article: R. Souillard, C. Woudstra, C. Le Maréchal, M. Dia, M. H. Bayon-Auboyer, M. Chemaly, P. Fach & S. Le Bouquin (2014) Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013, Avian Pathology, 43:5, 458-464, DOI: 10.1080/03079457.2014.957644 To link to this article: http://dx.doi.org/10.1080/03079457.2014.957644
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Avian Pathology, 2014 Vol. 43, No. 5, 458–464, http://dx.doi.org/10.1080/03079457.2014.957644
ORIGINAL ARTICLE
Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013 R. Souillard1*, C. Woudstra2, C. Le Maréchal3, M. Dia1, M. H. Bayon-Auboyer4, M. Chemaly3, P. Fach2, and S. Le Bouquin1 Anses – UEB, Ploufragan-Plouzané Laboratory, Avian and Rabbit Epidemiology and Welfare Unit, Ploufragan, France, Anses, Food Safety Laboratory, Maisons Alfort, France, 3Anses – UEB, Ploufragan-Plouzané Laboratory, Hygiene and Quality of Poultry and Pig Products Unit, Ploufragan, France, and 4Analysis and Development Laboratory 22 – LDA22/ Labocea, Ploufragan, France
1
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2
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.
Introduction Botulism is a severe flaccid paralytic disease caused by botulinum neurotoxins (BoNTs). BoNTs are produced by the anaerobic, spore-forming bacterium Clostridium botulinum and some strains of Clostridium baratii and Clostridium butyricum. Human disease has been mainly associated with BoNT types A, B, E and (more rarely) F, whereas animal botulism is mainly associated with BoNT types C and D (Dohms, 2008), although other BoNT types can sporadically be involved in animal botulism; for example, type A in equine outbreaks (Ostrowski et al., 2012) or type E in wild bird outbreaks (Brand et al., 1988). Mosaic toxins C–D or D–C have recently been associated with animal botulism, mainly represented in birds and cattle respectively (Skarin et al., 2010; Woudstra et al., 2012). Botulism was first reported in chickens in 1917 (Dickson, 1917) and has been reported many times in the literature since the 1970s. Avian botulism is a serious problem in European countries leading to significant economic losses (Lindberg et al., 2010). The number of botulism outbreaks has been increasing in several European countries during the past decade (Skarin et al., 2010, 2013). The factors behind this upsurge of avian botulism have not been identified and currently little is known about the epidemiology of the disease. C. botulinum spores can actually survive for decades in the environment and botulism can be recurrent in affected farms
(Okamoto et al., 1999). But no data are available regarding contamination by C. botulinum spores of poultry houses and surroundings after a botulism poultry outbreak. Such data are crucial for preventing recurrence of botulism in affected farms and preventing cross-contamination between farms. Detection of C. botulinum in the farm environment is of critical importance for the implementation of risk prevention and control of sanitary safety. The aim of this study was to compile descriptive epidemiological data and to characterize the contamination of C. botulinum within and around poultry houses after a botulism outbreak. For this purpose, 17 affected farms were investigated in France and contamination was evaluated by real-time polymerase chain reaction (PCR). Materials and Methods Sample collection from poultry farms. From 2011 to 2013, 17 poultry flocks associated with botulism outbreaks were investigated (Table 1). Outbreaks were reported by veterinarians and farm selection was based on the owners agreeing to take part in the study. Very few farmers refused to participate, essentially because of a lack of availability. Veterinarians in poultry farming reported clinical botulism based on typical signs such as flaccid paralysis and high mortality, and sent samples collected from birds (sera, intestinal contents and/or organs) to one of the three French laboratories allowed to perform analyses for diagnostic confirmation. Serum and/or intestinal content were analysed with the mouse lethality test to demonstrate the presence of BoNTs while intestinal content and/or organs
*To whom correspondence should be addressed: Tel: +33 2 96 01 62 22. Fax: +33 2 96 01 62 23. E-mail: [email protected] (Received 17 February 2014; accepted 31 July 2014) © 2014 Houghton Trust Ltd
C. botulinum in poultry farms in France 459 Table 1.
Farm no. 1
Poultry
2 3
February 2011 March 2011 April 2011
Broilers Turkeys
4 5 6
May 2011 June 2011 August 2011
Broilers Broilers Broilers
7
September 2011 May 2012
Broilers
8 9 10 11
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Date
12 13 14 15 16 17
July 2012 August 2012 September 2012 September 2012 October 2012 October 2012 February 2013 March 2013 May 2013
Turkeys
Ducks Broilers Broilers Ducks Turkeys Broilers Layers Turkeys Broilers Turkeys
Description of the 17 poultry flocks affected with botulism.
Numbers and sex
Sexes affected
5040 M 4480 F 22440 4204 M 3672 F 21420 26520 10200 M 10300 F 21930
F
77
14.1
Euthanasia (84)
– M
10 98
26.7 35.2
Euthanasia (21) Anticipated slaughter (109)
– – M
45 10 44
2.8 ND 7.8
Slaughter (56) Euthanasia (18) Slaughter (51)
–
31
11.2
Slaughter (50)
M
50
9.1
Slaughter (80)
45 30 M 78 F 42 110
10.0 8.7 M 5.6 F 16 17.8
35 ND M 76 F 67 ND 105
6.3 ND M 16.8 F 30.0 ND 4.5
11000 M 6000 F 30000 29700 8850 M 6250 F 4500 M 4400 F 20800 ND 4160 M 4000 F ND 5500 M
– – M&F M – ND M and F ND M
Age (days)
Mortality rate (%)
Fate of affected flock (age in days)
Slaughter (55) Euthanasia (35) M: slaughter (87) F: anticipated slaughter (60) Slaughter (120) Slaughter (44) ND M & F: anticipated slaughter (92) ND Slaughter (112)
M, males; F, females; ‘–‘, no sexed poultry; ND, no data. were analysed by PCR after an enrichment step in anaerobic conditions to identify the BoNT type. All investigated outbreaks were identified as type C or type C–D botulism, depending on the PCR target used by the laboratory to identify the toxin type. Samples were collected according to seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoir, water, and feed. Swabs on the ground, swabs on the walls, litter, soil, water, feed, darkling beetles (Alphitobius diaperinus), rodents and wild bird droppings were sampled. The 17 affected farms were visited to collect samples in the presence of poultry or just after their departure. For eight poultry farms, it was possible to make a second visit to collect samples after cleaning and disinfection operations. In each poultry house, swabs were attached to the shoes for a walk back and forth on the litter in each half of the house. Litter was collected randomly in a 500 ml container in five locations in the house. Soil was collected randomly in a 200 ml container from two locations. For the walls, a surface of 12 m2 was swabbed (6 × 2 m2) about 50 cm above the ground. In the changing room, 4 m2 (4 × 1 m2) of the walls were swabbed about 50 cm above the floor and swabs were also attached to the shoes for a walk back and forth in each half of the room. For the ventilation system, an area of 6 m2 was swabbed on the air admission and the air exit (6 × 1 m2). Water (500 ml) was sampled from the water lines, from the tanks and from the wells. Feed from plates, silo and hopper was sampled in a 500 ml container. For the surroundings, swabs were attached to the shoes for a walk around the outside of the poultry house. Soil was collected randomly in front of the house in a 200 ml container from two locations less than 1 m from the house. Swabs were also taken from a puddle. Concerning the animal reservoir, darkling beetles were collected in the houses in the litter under the plates or along the walls. A swab was also taken from the surface of a rodent carcass found outside a house. Wild bird or rodent droppings were also collected. The swabs used in this study were dry swabs without any specific transport medium.
Enrichment conditions and DNA extraction. Samples were diluted 1/10 in pre-reduced trypticase–peptone–glucose–yeast extract broth and cultured at 37 ± 1°C for 4 days under anaerobic conditions using an anaerobic chamber (10% hydrogen, 10% carbon dioxide, 80% nitrogen). An amount
of at least 20 g for feed, litter, soil or droppings, of at least 100 ml for water samples and from 10 to 15 darkling beetles (washed with detergent, rinsed and crushed) was analysed. The washing step was performed to ensure that any C. botulinum detected was present inside the beetles. For swabs, trypticase–peptone–glucose–yeast extract broth was added directly in the carrying bag so as to immerse the swab (from 200 to 250 ml). After incubation, 1 ml of each enrichment broth was collected. Cells were pelleted by centrifugation (6000 × g, 10 min) and subjected to DNA extraction using either a DNeasy blood and tissue kit or a QIAamp® DNA Mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions.
Real-time polymerase chain reaction. Real-time PCR, primers and probes used in this study were described previously (Woudstra et al., 2012). Either GeneDisc array “GD2 C, D & mosaic” with the V2 GeneDisc® cycler from Pall GeneDisc® (Bruz, France) Technologies, or real-time PCR using a BioRad CFX96 thermal cycler (Bio-Rad Laboratories, Marnes-la-Coquette, France) were used for detection of C. botulinum types C, C–D, D and D–C (Woudstra et al., 2012). Results and Cq (Quantification Cycle) values were recorded according to the GeneDisc® cycler or Bio-Rad CFX96 software applications. Concerning real-time PCR with the Bio-Rad CFX96 thermal cycler, each assay was performed in a total volume of 25 µl, containing 3 µl DNA template, 10 µl IQ supermix (Bio-Rad) and a final concentration of 600 nM for primers and 400 nM for probes. The thermal profile consisted of 5 min at 95°C, followed by 40 cycles of denaturing at 95°C for 15 sec and annealing extension at 55°C for 30 sec. Each run included positive and negative controls for each target and a commercial internal control (QuantiFast Pathogen + IC Kits; Qiagen) used according to manufacturer’s instructions. For both methods, a sample was considered positive when the Cq (Quantification Cycle) was below 35.
Results Poultry flocks affected by botulism. An epidemiological description of poultry flocks investigated between 2011 and 2013 is presented in Table 1. Most cases occurred between
460
R. Souillard et al.
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May and October (12/17 affected flocks). These outbreaks took place mainly in North-West France in 10 poultry organizations. Four productions were affected, essentially meat turkeys and broilers, but also two duck flocks (recurrent cases in the same house) and one layer flock. Flaccid paralysis was reported in all flocks. Cases of botulism in broilers occurred as early as 10 days old, whereas onset in turkeys, ducks and layers was later. Among the eight sexed poultry flocks, only males were affected in five houses. High mortalities were reported. The average mortality was 13.9% (standard deviation: 9.4%) and the median was 10.6%. The affected birds were usually treated with amoxicillin, but the data collected in our study did not provide enough accurate information to make conclusions concerning the treatment protocols and their effectiveness. For five out of the 15 investigated farms, this was not the first botulism outbreak. Cattle were present in 11 farms and remained healthy. Detection of C. botulinum in affected poultry farms. Between two and 18 samples were collected in each affected poultry farm. The 17 farms were visited either while poultry were present or just after their departure, and 39.5% (73/185) of samples tested positive (Table 2). All positive samples were recorded as C. botulinum type C–D, except one swab sampled on the wall of a changing room (Flock No. 13) recorded as type D–C. Four areas were positive in more than one-half of affected farms (Figure 1): animal reservoir with darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). Among the 14 samples collected from possible animal reservoirs, darkling beetles provided the most positive results, and one swab from the surface of a rodent carcass was also positive. In the houses, 48.8% (21/43) of samples were positive with swabs on the litter, swabs on the walls and litter samples. The litter was straw and/or wood chips, except for the ducks which were on slatted floors. No link was observed between litter composition and the detection of C. botulinum. Regarding water contamination, 40% (14/ 35) of water samples were positive both in the distribution system and in wells. In terms of the surroundings, C. botulinum type C–D was identified in 55.1% (16/29) of samples, essentially in swabs on the soil and in soil samples. C. botulinum type C–D was also detected in a puddle near a poultry house. In seven changing rooms out of 15 poultry farms, C. botulinum was detected with swabs on the ground and on the walls. Concerning the ventilation system, C. botulinum type C–D was detected in four out of nine poultry houses with swabs in air admission and in air exit vents. In the various tests for feed contamination, only one feed plate was positive. On eight occasions, poultry farms were sampled after cleaning and disinfection. Between three and 15 samples were collected in each house. A total 12.7% (8/63) of samples were still positive after these operations (Table 3). Contamination was detected in four out of the eight farms investigated. The ventilation system returned positive results in three farms. In the houses, only one swab on the soil was positive. Regarding the surroundings, positive samples were reported in two farms. Collected data in our study did not provide enough accurate information to make a link between the cleaning and disinfection procedures and the persistence of C. botulinum.
Discussion Estimation of botulism prevalence in poultry flocks is difficult. This difficulty can be explained by the fact that it is not a notifiable disease in all European countries and because diagnoses based on clinical signs are also sometimes difficult to confirm by laboratory investigations (Skarin et al., 2013). In this study, a relatively high number of botulism outbreaks in poultry farms were investigated in comparison with reported cases in the literature. Only one other study reported such a large number of botulism outbreaks, 30 years ago (27 outbreaks in 16 farms) (Dohms et al., 1982). Botulism outbreaks have already been reported many times in broiler flocks (Roberts & Collings, 1973; Dohms et al., 1982; Pecelunas et al., 1999), but more rarely in other poultry productions such as turkeys (Smart et al., 1983; Popp et al., 2012), layers (Sharpe et al., 2011) and ducks, in which wildlife outbreaks accounted for most of the cases reported (Dohms, 2008). Descriptive epidemiological data obtained from this study concur with previous reports. Botulism has a seasonal incidence; the disease in poultry occurs more frequently during warmer months (Dohms, 2008).This was confirmed here, as most botulism outbreaks investigated occurred between May and October. Most cases of botulism in chickens have actually been reported to occur between 2 and 8 weeks of age (Dohms, 2008), although the disease has been reported at later stages of life (Trampel et al., 2005). Here we found that broiler outbreaks occurred between 10 and 45 days old. On the contrary, turkeys were much older, between 67 and 110 days old, in accordance with previous reports (Smart et al., 1983; Popp et al., 2012). Males seemed to be more affected by botulism than females. This was also reported in other turkey botulism outbreaks where only toms were affected (Smart et al., 1983; Popp et al., 2012). However, the factors behind this phenomenon remain unknown. In our study, mortality ranged from about 2.8% to 35.2%. Mortality rates typically range from 1% to 25% in poultry flocks (Dohms et al., 1982) but higher mortality was reported, up to 30% (Smart et al., 1983) or 50% in turkeys (Popp et al., 2012) and 84% in pheasants (Blandford, 1976). Despite causing heavy financial losses, little is yet known of the epidemiology of the disease, notably contamination during a poultry botulism outbreak. The traditional method for detection and identification of C. botulinum in samples is enrichment of anaerobic culture followed by a mouse bioassay. However, this method is not appropriate for running a large number of samples, and alternatives such as PCR methods allow easier testing of large numbers (Lindberg et al., 2010). Moreover, real-time PCR has proved to be a sensitive tool for the detection of C. botulinum in the environment and for studying its ecoepidemiology (Vidal et al., 2013). In previous studies, it has been demonstrated that such an approach gives similar results to the mouse bioassay—100% agreement with Franciosa et al. (1996) and Hielm et al. (1996), 88.9% in Fach et al. (2002)— and that it provides a good alternative to conventional methodologies. It has even been shown that such a PCR-based approach with an enrichment step is more sensitive than the mouse bioassay for detecting the presence of C. botulinum in naturally contaminated samples (Fach et al., 2002). In our study, we thus used a real-time PCR approach combined with an enrichment step to detect the presence of C. botulinum in the environment of poultry flocks. The enrichment step is required to enable the
Table 2. C. botulinum type C–D detection in each affected farm in the presence of poultry or shortly after their departure.
Farm number
Changing room Ventilation Water
Feed
Surroundings
Animal reservoir
Total
Swab on the litter Litter Soil Swab on the walls Swab on the walls Swab on the ground
2
3
4
5
6
7
8
9
10
11
E
P
P
P
E
E
P
P
P
E
E
1/1
1/1 1/1
0/1 0/1
1/1 0/1
1/1 1/1
1/1 1/1
1/1 1/1 2/2
1/1
0/1
0/1
0/1 1/1
1/2 0/1
1/1
0/1
1/1
0/1
1/1
1/1
Swab in air admission Swab in air exit 1/1
Feed plate Feed silo Feed hopper
0/1
Swab on the soil Soil Swab in puddle
0/1
1/1
0/1
1/1
1/1
1/1
1/1
0/2 0/2
1/1
1/1
1/1 0/1 1/4
Darkling beetles Swab on rodent carcass Rodents droppings Wild birds droppings
0/1 0/1 0/1
0/1 0/1
1/1
0/1
0/1
0/1
0/1
0/1
1/1 0/1 0/1
1/1
1/1
0/1
1/1
0/1 0/1
0/1
0/1 0/1 0/1
1/1
1/1
1/2
1/1
1/1 0/1
13
14
15
16
17
P
E
P
P
P
E
0/1 0/2 0/1 0/1
1/1 0/1 0/1 0/1
0/1
b
1/1
0/1 1/1 0/1
1/1
1/1
0/1 2/2
0/2
0/2
0/1
0/1
1/1 1/2
Water pipeline Water tank Well water
0/1
0/1
a
12
1/1
0/1 1/1
1/1
0/1 1/1
0/1
0/2
0/2
1/2
0/2
1/2
0/2
0/2 2/3 1/1 3/4
0/1 0/1
1/1
0/1
0/1 0/1
1/1
1/1
0/1 1/2
1/1 1/1 0/1
P: poultry; E: empty; aSlatted floors. bC.botulinum type D–C.
5/9
0/8
4/7
6/9
6/8
6/9
5/15
6/9
10/18
2/17
2/16
6/15
2/8 5/8
7/16
3/11 1/4
4/15
2/2
1/2
12/22 0/3 2/10
14/35
1/8 0/11 0/6
1/25
11/19 2/6 3/4
16/29
9/11 1/1 0/1 0/1
10/14
0/1 1/1 0/1
0/1 0/1 0/2
1/1
3/10
21/43
0/2 0/2
1/1
0/1 2/5
9/16 6/11 0/4 6/12
0/1 0/1
0/1 0/1 1/1
Total positive samples
7/11
1/2
2/17
73/185
C. botulinum in poultry farms in France 461
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House
1
R. Souillard et al.
Number of farms
462
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
Negave Posive
*
**
Poultry house
*
**
*
Changing room
**
*
Venlaon
**
*
Surroundings
**
*
Water
*
Feed Darkling beetles
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Figure 1. Detection of C. botulinum by real-time PCR in 17 affected poultry farms. *In the presence of poultry or shortly after their departure. **Post cleaning and disinfection.
Table 3. C. botulinum type C–D detection in each affected farm post cleaning and disinfection.
Farm number 8 House
Swab on the soil Soil Swab on the walls
9 0/2
0/2
0/1
0/1
0/2
0/1
Water
Water pipeline
Changing room
Swab on the walls
0/1
Ventilation
Swab in air admission Swab in air exit
1/1
Swab on the soil Soil Swab in puddle
1/1
Surroundings
Total a
10
a
11 0/1
a
12
13
15
16
0/1 0/1 0/1
0/1 0/1 0/1
0/2 0/2 0/2
1/1 0/2 0/1
0/2
0/2
0/1
0/1
0/1
0/1 0/1 1/1 0/1
0/1
0/1 0/1
0/2
0/1 0/1 0/1
0/1 0/2
1/11 0/6 0/9
1/26
0/5
0/5
0/1
0/5
0/5
1/1 2/2
0/1
3/8 2/3
5/11
0/1 1/2
0/1 0/2
1/8 1/7 0/1
2/16
0/1 2/5
1/6
0/10
0/3
0/6
0/10
Total positive samples
4/15
1/9
8/63
Slatted floors.
detection of low concentrations of C. botulinum (Vidal et al., 2011). This also ensures that only viable bacteria are detected, as dead bacteria or residual DNA are diluted in the enrichment broth, thus reducing the probability of detecting them during the subsequent PCR assay. Such an approach has recently been successfully used to develop a mostprobable-number PCR (MPN PCR) method to detect type E C. botulinum in samples collected from beaches on Lakes Michigan and Ontario and to demonstrate the presence of vegetative cells and not spore cells. In this study, all heattreated samples were negative for the bont/E gene whereas they were positive without heat treatment after enrichment, demonstrating that only viable cells were detected by PCR despite the presence of a small number of dead cells in the enrichment (Chun et al., 2013). In our study, to explore a large number of areas, we tested 21 different types of samples as appropriate for the specific circumstances of each farm, which explains the variability of sampling plan in each farm (from two to 18 samples) and the relatively low number of samples in each area (from 14 to 43 samples). The presence of C. botulinum type C–D in the environment of affected farms was detected in 39.5% of
the samples. C. botulinum was also detected after cleaning and disinfection of the poultry houses, indicating the difficulties of decontamination operations. This is illustrated by the number of recurrences among the investigated flocks and has been reported in previous studies (Okamoto et al., 1999). C. botulinum is considered ubiquitous. However, recent studies conducted in Norway and France in 46 farms without previously reported outbreaks (Hardy & Kaldhusdal, 2013; Souillard et al., 2013) showed that type C–D C. botulinum is not frequent in healthy poultry farms (only three positive samples in one out of the 46 farms investigated). This therefore indicates that positive samples detected in our study do not result from the normal presence of type C–D C. botulinum but are linked to botulism outbreaks. C. botulinum type C–D was detected in all positive samples, except in one swab in a changing room that was positive for type D–C. C. botulinum type C–D is clearly the most prevalent BoNT type associated with avian botulism at present in Europe (Woudstra et al., 2012). A study including different European countries showed that avian botulism is mostly associated with type C–D whereas
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C. botulinum in poultry farms in France 463
bovine botulism is associated with type D–C. However, in a few cases both types (C–D and D–C) were detected in turkeys, pheasants and wild ducks (Woudstra et al., 2012). Consequently, the type D–C detected in the changing room could be related to the botulism event in the poultry house. It is unknown whether the mosaic types are emerging or whether they are now being diagnosed because of the development of new methods that allow the discrimination of mosaic from non-mosaic types. In our study, C. botulinum type C–D was frequently detected in the litter. Litter has often been reported as being contaminated during botulism outbreaks. In a similar study, C. botulinum type C was detected in 26 out of 38 litter samples collected in affected broiler farms (Dohms et al., 1982). In another broiler outbreak, soil and litter were also reported as contaminated (Okamoto et al., 1999). Litter inevitably becomes contaminated with droppings and poultry carcasses. Dust essentially composed of contaminated droppings was also found to be positive with swabs from the walls of the houses. Spores of C. botulinum may be transferred from the affected houses to several areas. Indeed, we detected C. botulinum in the changing rooms, in the ventilation system and in the surroundings of the houses. C. botulinum may have been transported into the changing room via contaminated shoes or equipment. In the ventilation system, C. botulinum may have been disseminated by dust, which was found to be contaminated when sampled with swabs. Cleaning and disinfection of an air admission and exit circuit is not easy, because of the difficulty of accessing the system; this could explain the persistence of the spores after these operations. The surroundings of the houses were also frequently contaminated probably via farming activities (movement of the staff and the farmer, use of farm equipment) or via dust from the air exit system. Spores of C. botulinum are soil-associated and resistant in the environment. It has also been reported previously that mud or soil was contaminated by C. botulinum type C during outbreaks in wild birds (Franciosa et al., 1996) and in affected broiler farms (Okamoto et al., 1999). In our study, C. botulinum was found to have persisted in the soil inside the house and in the surroundings after decontamination operations. It appears to be difficult to ensure the complete destruction of spore forms in soil (Dohms, 2008), so this is an important factor to be addressed to prevent recurrence of the disease. Concerning animal reservoirs, invertebrates, notably fly maggots, are well-known vehicles of BoNTs during outbreaks of water bird botulism (Hubálek & Halouzka, 1991). C. botulinum type C was also recently observed in adult Calliphoridae and Sarcophagida flies collected around bird carcasses during a botulism outbreak (Vidal et al., 2013). In our study, darkling beetles were also found to be frequently positive. This suggests that darkling beetles could be vectors of C. botulinum and a reservoir of the bacteria. These beetles feed on bird droppings, bird carcasses or components of the litter, which could explain why these invertebrates have been shown to harbour C. botulinum (Hinkle & Hickle, 2008). Poultry eat darkling beetles, a natural source of food, and can thus become infected. Darkling beetles have already been shown to harbour several avian pathogens such as Escherichia coli, Salmonella spp. and Campylobacter spp. (Hinkle & Hickle, 2008). Beetles can actually be involved in Salmonella enterica transmission between two consecutive broiler flocks (Skov et al., 2004) and S. enterica can transit in the gut of darkling beetles, inducing a dispersion of viable pathogenic bacteria
within 2 to 3 h (Zheng et al., 2012). Darkling beetles are abundant in poultry production and difficult to suppress. Disinfestation is thus crucial in handling botulism outbreaks and preventing recurrence. Rodents are also a reservoir of a variety of pathogens (Hinkle & Hickle, 2008) and notably of C. botulinum (Popoff, 1995). Here one swab collected from a rodent carcass was found to be positive. Whether the rodent carcass was the source of the outbreak or the result of cross-contamination was not determined. In a turkey botulism outbreak, C. botulinum was also isolated from the liver of a dead rat found close to the barn (Popp et al., 2012). This illustrates how important the management of rodents is to prevent dissemination of the disease. Water, frequently found to be positive in our study, has already been suspected as a source of contamination during a bird outbreak in a zoo (Raymundo et al., 2012) and C. botulinum has been detected in mud samples obtained from different raw-water storage areas that were affected by waterfowl botulism in the Netherlands (Notermans et al., 1980), but few data are available concerning water contamination in poultry farms. In a recent case report, no water sample, taken from pipes and Plasson (bell) drinkers, was positive (Popp et al., 2012). In our study, water samples from the distribution system may have been contaminated by dust in the tank or by biofilm in the pipelines. The hydrophobicity of C. botulinum spores could enable them to adhere to surfaces and biofilm formation (Wiencek et al., 1990). Moreover, two well-water samples were positive. This well water is used as drinking water and could also explain the contamination of water pipeline samples. The well water could be contaminated by spores of C. botulinum, with carcasses of rodents or other small animals. Further investigations are needed to better understand the role of drinking water as a source of contamination in poultry farms. Finally, only one feed plate was positive, probably contaminated by droppings or dust. It is difficult to detect C. botulinum in feed, probably because of the sampling method. Few reports have mentioned it, but feed could be a source of contamination (Dohms et al., 1982). This study identifies the critical contaminated areas in poultry farms after a botulism outbreak. Darkling beetles, drinking water, the ventilation system and soil from the surroundings and the houses are the main contaminated areas that have to be taken into account for handling botulism outbreaks. These findings are essential for implementing control of the disease and will enable us to assess a decontamination procedure to prevent the recurrence of avian botulism.
Acknowledgements The authors are grateful to the poultry farmers, veterinarians and technicians. They would like to thank Denis Léon, Sandra Rouxel, Emmanuelle Houard and Valéria Guimaraes for their participation in the study.
Funding This project was made possible by financial support from the French Ministry of Agriculture and the French Agency for Food, Environmental and Occupational Health & Safety.
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Popoff, M.R. (1995). Ecology of neurotoxigenic strains of clostridia. In C. Montecucco (Ed.). Clostridial Neurotoxins (pp. 1–29). Berlin: SpringerVerlag. Popp, C., Hauck, R., Gad, W. & Hafez, H.M. (2012). Type C botulism in a commercial turkey farm: a case report. Avian Diseases, 56, 760–763. Raymundo, D.L., Von Hohendorf, R., Boabaid, F.M., Both, M.C., Sonne, L., Assis, R.A., Caldas, R.P. & Driemeier, D. (2012). Outbreak of type C botulism in captive wild birds. Journal of Zoo and Wildlife Medicine, 43, 388–390. Roberts, T.A. & Collings, D.F. (1973). An outbreak of type C botulism in broiler chicken. Avian Diseases, 17, 650–658. Sharpe, A.E., Sharpe, E.J., Ryan, E.D., Clarke, H.J. & McGettrick, S.A. (2011). Outbreak of type C botulism in laying hens. The Veterinary Record, 168, 669. Skarin, H., Lindberg, A., Blomqvist, G., Aspán, A. & Båverud, V. (2010). Molecular characterization and comparison of Clostridium botulinum type C avian strains. Avian Pathology, 39, 511–518. Skarin, H., Tevell Åberg, A., Woudstra, C., Hansen, T., Löfström, C., Koene, M., Bano, L., Hedeland, M., Anniballi, F., De Medici, D. & Olsson Engvall, E. (2013). The workshop on animal botulism in Europe. Biosecurity and Bioterrorism, 11, S183–S190. Skov, M.N., Spencer, A.G., Hald, B., Petersen, L., Nauerby, B., Carstensen, B. & Madsen, M. (2004). The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks. Avian Diseases, 48, 9–18. Smart, J.L., Laing, P.W. & Winkler, C.E. (1983). Type C botulism in intensively farmed turkeys. The Veterinary Record, 113, 198–200. Souillard, R., Woudstra, C., Dia, M., Léon, D., Toux, J.Y., Guimaraes, V., Bayon-Auboyer, M.H., Michel, V., Le Bouquin, S. & Fach, P. (2013). Uses of real-time PCR assay for detection of Clostridium botulinum neurotoxinogen in diseased and healthy poultry farms. In 10ème Journées de la Recherche Avicole et Palmipèdes à Foie Gras (pp. 341– 345). La Rochelle. Trampel, D.W., Smith, S.R. & Rocke, T.E. (2005). Toxicoinfectious botulism in commercial caponized chickens. Avian Diseases, 49, 301–303. Vidal, D., Anza, I., Taggart, M.A., Pérez-ramírez, E., Crespo, E., Hofle, U. & Mateo, R. (2013). Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands. Applied and Environmental Microbiology, 79, 4264–4271. Vidal, D., Taggart, M.A., Badiola, I. & Mateo, R. (2011). Real-time polymerase chain reaction for the detection of toxigenic Clostridium botulinum type C1 in waterbird and sediment samples: comparison with other PCR techniques. Journal of Veterinary Diagnostic Investigation, 23, 942–946. Wiencek, K.M., Klapes, N.A. & Foegeding, P.M. (1990). Hydrophobicity of Bacillus and Clostridium spores. Applied and Environmental Microbiology, 56, 2600–2605. Woudstra, C., Skarin, H., Anniballi, F., Fenicia, L., Bano, L., Drigo, I., Koene, M., Bäyon-Auboyer, M.H., Buffereau, J.P., De Medici, D. & Fach, P. (2012). Neurotoxin gene profiling of Clostridium botulinum types C and D native to different countries within Europe. Applied and Environmental Microbiology, 78, 3120–3127. Zheng, L., Crippen, T.L., Sheffield, C.L., Poole, T.L., Yu, Z. & Tomberlin, J.K. (2012). Evaluation of Salmonella movement through the gut of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Vector-Borne and Zoonotic Diseases, 12, 287–292.
Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013 a
b
c
a
d
R. Souillard , C. Woudstra , C. Le Maréchal , M. Dia , M. H. Bayon-Auboyer , M. c
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a
Chemaly , P. Fach & S. Le Bouquin a
Anses – UEB, Ploufragan-Plouzané Laboratory, Avian and Rabbit Epidemiology and Welfare Unit, Ploufragan, France b
Anses, Food Safety Laboratory, Maisons Alfort, France
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Anses – UEB, Ploufragan-Plouzané Laboratory, Hygiene and Quality of Poultry and Pig Products Unit, Ploufragan, France d
Analysis and Development Laboratory 22 – LDA22/Labocea, Ploufragan, France Accepted author version posted online: 30 Aug 2014.Published online: 21 Oct 2014.
Click for updates To cite this article: R. Souillard, C. Woudstra, C. Le Maréchal, M. Dia, M. H. Bayon-Auboyer, M. Chemaly, P. Fach & S. Le Bouquin (2014) Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013, Avian Pathology, 43:5, 458-464, DOI: 10.1080/03079457.2014.957644 To link to this article: http://dx.doi.org/10.1080/03079457.2014.957644
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Avian Pathology, 2014 Vol. 43, No. 5, 458–464, http://dx.doi.org/10.1080/03079457.2014.957644
ORIGINAL ARTICLE
Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013 R. Souillard1*, C. Woudstra2, C. Le Maréchal3, M. Dia1, M. H. Bayon-Auboyer4, M. Chemaly3, P. Fach2, and S. Le Bouquin1 Anses – UEB, Ploufragan-Plouzané Laboratory, Avian and Rabbit Epidemiology and Welfare Unit, Ploufragan, France, Anses, Food Safety Laboratory, Maisons Alfort, France, 3Anses – UEB, Ploufragan-Plouzané Laboratory, Hygiene and Quality of Poultry and Pig Products Unit, Ploufragan, France, and 4Analysis and Development Laboratory 22 – LDA22/ Labocea, Ploufragan, France
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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.
Introduction Botulism is a severe flaccid paralytic disease caused by botulinum neurotoxins (BoNTs). BoNTs are produced by the anaerobic, spore-forming bacterium Clostridium botulinum and some strains of Clostridium baratii and Clostridium butyricum. Human disease has been mainly associated with BoNT types A, B, E and (more rarely) F, whereas animal botulism is mainly associated with BoNT types C and D (Dohms, 2008), although other BoNT types can sporadically be involved in animal botulism; for example, type A in equine outbreaks (Ostrowski et al., 2012) or type E in wild bird outbreaks (Brand et al., 1988). Mosaic toxins C–D or D–C have recently been associated with animal botulism, mainly represented in birds and cattle respectively (Skarin et al., 2010; Woudstra et al., 2012). Botulism was first reported in chickens in 1917 (Dickson, 1917) and has been reported many times in the literature since the 1970s. Avian botulism is a serious problem in European countries leading to significant economic losses (Lindberg et al., 2010). The number of botulism outbreaks has been increasing in several European countries during the past decade (Skarin et al., 2010, 2013). The factors behind this upsurge of avian botulism have not been identified and currently little is known about the epidemiology of the disease. C. botulinum spores can actually survive for decades in the environment and botulism can be recurrent in affected farms
(Okamoto et al., 1999). But no data are available regarding contamination by C. botulinum spores of poultry houses and surroundings after a botulism poultry outbreak. Such data are crucial for preventing recurrence of botulism in affected farms and preventing cross-contamination between farms. Detection of C. botulinum in the farm environment is of critical importance for the implementation of risk prevention and control of sanitary safety. The aim of this study was to compile descriptive epidemiological data and to characterize the contamination of C. botulinum within and around poultry houses after a botulism outbreak. For this purpose, 17 affected farms were investigated in France and contamination was evaluated by real-time polymerase chain reaction (PCR). Materials and Methods Sample collection from poultry farms. From 2011 to 2013, 17 poultry flocks associated with botulism outbreaks were investigated (Table 1). Outbreaks were reported by veterinarians and farm selection was based on the owners agreeing to take part in the study. Very few farmers refused to participate, essentially because of a lack of availability. Veterinarians in poultry farming reported clinical botulism based on typical signs such as flaccid paralysis and high mortality, and sent samples collected from birds (sera, intestinal contents and/or organs) to one of the three French laboratories allowed to perform analyses for diagnostic confirmation. Serum and/or intestinal content were analysed with the mouse lethality test to demonstrate the presence of BoNTs while intestinal content and/or organs
*To whom correspondence should be addressed: Tel: +33 2 96 01 62 22. Fax: +33 2 96 01 62 23. E-mail: [email protected] (Received 17 February 2014; accepted 31 July 2014) © 2014 Houghton Trust Ltd
C. botulinum in poultry farms in France 459 Table 1.
Farm no. 1
Poultry
2 3
February 2011 March 2011 April 2011
Broilers Turkeys
4 5 6
May 2011 June 2011 August 2011
Broilers Broilers Broilers
7
September 2011 May 2012
Broilers
8 9 10 11
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Date
12 13 14 15 16 17
July 2012 August 2012 September 2012 September 2012 October 2012 October 2012 February 2013 March 2013 May 2013
Turkeys
Ducks Broilers Broilers Ducks Turkeys Broilers Layers Turkeys Broilers Turkeys
Description of the 17 poultry flocks affected with botulism.
Numbers and sex
Sexes affected
5040 M 4480 F 22440 4204 M 3672 F 21420 26520 10200 M 10300 F 21930
F
77
14.1
Euthanasia (84)
– M
10 98
26.7 35.2
Euthanasia (21) Anticipated slaughter (109)
– – M
45 10 44
2.8 ND 7.8
Slaughter (56) Euthanasia (18) Slaughter (51)
–
31
11.2
Slaughter (50)
M
50
9.1
Slaughter (80)
45 30 M 78 F 42 110
10.0 8.7 M 5.6 F 16 17.8
35 ND M 76 F 67 ND 105
6.3 ND M 16.8 F 30.0 ND 4.5
11000 M 6000 F 30000 29700 8850 M 6250 F 4500 M 4400 F 20800 ND 4160 M 4000 F ND 5500 M
– – M&F M – ND M and F ND M
Age (days)
Mortality rate (%)
Fate of affected flock (age in days)
Slaughter (55) Euthanasia (35) M: slaughter (87) F: anticipated slaughter (60) Slaughter (120) Slaughter (44) ND M & F: anticipated slaughter (92) ND Slaughter (112)
M, males; F, females; ‘–‘, no sexed poultry; ND, no data. were analysed by PCR after an enrichment step in anaerobic conditions to identify the BoNT type. All investigated outbreaks were identified as type C or type C–D botulism, depending on the PCR target used by the laboratory to identify the toxin type. Samples were collected according to seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoir, water, and feed. Swabs on the ground, swabs on the walls, litter, soil, water, feed, darkling beetles (Alphitobius diaperinus), rodents and wild bird droppings were sampled. The 17 affected farms were visited to collect samples in the presence of poultry or just after their departure. For eight poultry farms, it was possible to make a second visit to collect samples after cleaning and disinfection operations. In each poultry house, swabs were attached to the shoes for a walk back and forth on the litter in each half of the house. Litter was collected randomly in a 500 ml container in five locations in the house. Soil was collected randomly in a 200 ml container from two locations. For the walls, a surface of 12 m2 was swabbed (6 × 2 m2) about 50 cm above the ground. In the changing room, 4 m2 (4 × 1 m2) of the walls were swabbed about 50 cm above the floor and swabs were also attached to the shoes for a walk back and forth in each half of the room. For the ventilation system, an area of 6 m2 was swabbed on the air admission and the air exit (6 × 1 m2). Water (500 ml) was sampled from the water lines, from the tanks and from the wells. Feed from plates, silo and hopper was sampled in a 500 ml container. For the surroundings, swabs were attached to the shoes for a walk around the outside of the poultry house. Soil was collected randomly in front of the house in a 200 ml container from two locations less than 1 m from the house. Swabs were also taken from a puddle. Concerning the animal reservoir, darkling beetles were collected in the houses in the litter under the plates or along the walls. A swab was also taken from the surface of a rodent carcass found outside a house. Wild bird or rodent droppings were also collected. The swabs used in this study were dry swabs without any specific transport medium.
Enrichment conditions and DNA extraction. Samples were diluted 1/10 in pre-reduced trypticase–peptone–glucose–yeast extract broth and cultured at 37 ± 1°C for 4 days under anaerobic conditions using an anaerobic chamber (10% hydrogen, 10% carbon dioxide, 80% nitrogen). An amount
of at least 20 g for feed, litter, soil or droppings, of at least 100 ml for water samples and from 10 to 15 darkling beetles (washed with detergent, rinsed and crushed) was analysed. The washing step was performed to ensure that any C. botulinum detected was present inside the beetles. For swabs, trypticase–peptone–glucose–yeast extract broth was added directly in the carrying bag so as to immerse the swab (from 200 to 250 ml). After incubation, 1 ml of each enrichment broth was collected. Cells were pelleted by centrifugation (6000 × g, 10 min) and subjected to DNA extraction using either a DNeasy blood and tissue kit or a QIAamp® DNA Mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions.
Real-time polymerase chain reaction. Real-time PCR, primers and probes used in this study were described previously (Woudstra et al., 2012). Either GeneDisc array “GD2 C, D & mosaic” with the V2 GeneDisc® cycler from Pall GeneDisc® (Bruz, France) Technologies, or real-time PCR using a BioRad CFX96 thermal cycler (Bio-Rad Laboratories, Marnes-la-Coquette, France) were used for detection of C. botulinum types C, C–D, D and D–C (Woudstra et al., 2012). Results and Cq (Quantification Cycle) values were recorded according to the GeneDisc® cycler or Bio-Rad CFX96 software applications. Concerning real-time PCR with the Bio-Rad CFX96 thermal cycler, each assay was performed in a total volume of 25 µl, containing 3 µl DNA template, 10 µl IQ supermix (Bio-Rad) and a final concentration of 600 nM for primers and 400 nM for probes. The thermal profile consisted of 5 min at 95°C, followed by 40 cycles of denaturing at 95°C for 15 sec and annealing extension at 55°C for 30 sec. Each run included positive and negative controls for each target and a commercial internal control (QuantiFast Pathogen + IC Kits; Qiagen) used according to manufacturer’s instructions. For both methods, a sample was considered positive when the Cq (Quantification Cycle) was below 35.
Results Poultry flocks affected by botulism. An epidemiological description of poultry flocks investigated between 2011 and 2013 is presented in Table 1. Most cases occurred between
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May and October (12/17 affected flocks). These outbreaks took place mainly in North-West France in 10 poultry organizations. Four productions were affected, essentially meat turkeys and broilers, but also two duck flocks (recurrent cases in the same house) and one layer flock. Flaccid paralysis was reported in all flocks. Cases of botulism in broilers occurred as early as 10 days old, whereas onset in turkeys, ducks and layers was later. Among the eight sexed poultry flocks, only males were affected in five houses. High mortalities were reported. The average mortality was 13.9% (standard deviation: 9.4%) and the median was 10.6%. The affected birds were usually treated with amoxicillin, but the data collected in our study did not provide enough accurate information to make conclusions concerning the treatment protocols and their effectiveness. For five out of the 15 investigated farms, this was not the first botulism outbreak. Cattle were present in 11 farms and remained healthy. Detection of C. botulinum in affected poultry farms. Between two and 18 samples were collected in each affected poultry farm. The 17 farms were visited either while poultry were present or just after their departure, and 39.5% (73/185) of samples tested positive (Table 2). All positive samples were recorded as C. botulinum type C–D, except one swab sampled on the wall of a changing room (Flock No. 13) recorded as type D–C. Four areas were positive in more than one-half of affected farms (Figure 1): animal reservoir with darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). Among the 14 samples collected from possible animal reservoirs, darkling beetles provided the most positive results, and one swab from the surface of a rodent carcass was also positive. In the houses, 48.8% (21/43) of samples were positive with swabs on the litter, swabs on the walls and litter samples. The litter was straw and/or wood chips, except for the ducks which were on slatted floors. No link was observed between litter composition and the detection of C. botulinum. Regarding water contamination, 40% (14/ 35) of water samples were positive both in the distribution system and in wells. In terms of the surroundings, C. botulinum type C–D was identified in 55.1% (16/29) of samples, essentially in swabs on the soil and in soil samples. C. botulinum type C–D was also detected in a puddle near a poultry house. In seven changing rooms out of 15 poultry farms, C. botulinum was detected with swabs on the ground and on the walls. Concerning the ventilation system, C. botulinum type C–D was detected in four out of nine poultry houses with swabs in air admission and in air exit vents. In the various tests for feed contamination, only one feed plate was positive. On eight occasions, poultry farms were sampled after cleaning and disinfection. Between three and 15 samples were collected in each house. A total 12.7% (8/63) of samples were still positive after these operations (Table 3). Contamination was detected in four out of the eight farms investigated. The ventilation system returned positive results in three farms. In the houses, only one swab on the soil was positive. Regarding the surroundings, positive samples were reported in two farms. Collected data in our study did not provide enough accurate information to make a link between the cleaning and disinfection procedures and the persistence of C. botulinum.
Discussion Estimation of botulism prevalence in poultry flocks is difficult. This difficulty can be explained by the fact that it is not a notifiable disease in all European countries and because diagnoses based on clinical signs are also sometimes difficult to confirm by laboratory investigations (Skarin et al., 2013). In this study, a relatively high number of botulism outbreaks in poultry farms were investigated in comparison with reported cases in the literature. Only one other study reported such a large number of botulism outbreaks, 30 years ago (27 outbreaks in 16 farms) (Dohms et al., 1982). Botulism outbreaks have already been reported many times in broiler flocks (Roberts & Collings, 1973; Dohms et al., 1982; Pecelunas et al., 1999), but more rarely in other poultry productions such as turkeys (Smart et al., 1983; Popp et al., 2012), layers (Sharpe et al., 2011) and ducks, in which wildlife outbreaks accounted for most of the cases reported (Dohms, 2008). Descriptive epidemiological data obtained from this study concur with previous reports. Botulism has a seasonal incidence; the disease in poultry occurs more frequently during warmer months (Dohms, 2008).This was confirmed here, as most botulism outbreaks investigated occurred between May and October. Most cases of botulism in chickens have actually been reported to occur between 2 and 8 weeks of age (Dohms, 2008), although the disease has been reported at later stages of life (Trampel et al., 2005). Here we found that broiler outbreaks occurred between 10 and 45 days old. On the contrary, turkeys were much older, between 67 and 110 days old, in accordance with previous reports (Smart et al., 1983; Popp et al., 2012). Males seemed to be more affected by botulism than females. This was also reported in other turkey botulism outbreaks where only toms were affected (Smart et al., 1983; Popp et al., 2012). However, the factors behind this phenomenon remain unknown. In our study, mortality ranged from about 2.8% to 35.2%. Mortality rates typically range from 1% to 25% in poultry flocks (Dohms et al., 1982) but higher mortality was reported, up to 30% (Smart et al., 1983) or 50% in turkeys (Popp et al., 2012) and 84% in pheasants (Blandford, 1976). Despite causing heavy financial losses, little is yet known of the epidemiology of the disease, notably contamination during a poultry botulism outbreak. The traditional method for detection and identification of C. botulinum in samples is enrichment of anaerobic culture followed by a mouse bioassay. However, this method is not appropriate for running a large number of samples, and alternatives such as PCR methods allow easier testing of large numbers (Lindberg et al., 2010). Moreover, real-time PCR has proved to be a sensitive tool for the detection of C. botulinum in the environment and for studying its ecoepidemiology (Vidal et al., 2013). In previous studies, it has been demonstrated that such an approach gives similar results to the mouse bioassay—100% agreement with Franciosa et al. (1996) and Hielm et al. (1996), 88.9% in Fach et al. (2002)— and that it provides a good alternative to conventional methodologies. It has even been shown that such a PCR-based approach with an enrichment step is more sensitive than the mouse bioassay for detecting the presence of C. botulinum in naturally contaminated samples (Fach et al., 2002). In our study, we thus used a real-time PCR approach combined with an enrichment step to detect the presence of C. botulinum in the environment of poultry flocks. The enrichment step is required to enable the
Table 2. C. botulinum type C–D detection in each affected farm in the presence of poultry or shortly after their departure.
Farm number
Changing room Ventilation Water
Feed
Surroundings
Animal reservoir
Total
Swab on the litter Litter Soil Swab on the walls Swab on the walls Swab on the ground
2
3
4
5
6
7
8
9
10
11
E
P
P
P
E
E
P
P
P
E
E
1/1
1/1 1/1
0/1 0/1
1/1 0/1
1/1 1/1
1/1 1/1
1/1 1/1 2/2
1/1
0/1
0/1
0/1 1/1
1/2 0/1
1/1
0/1
1/1
0/1
1/1
1/1
Swab in air admission Swab in air exit 1/1
Feed plate Feed silo Feed hopper
0/1
Swab on the soil Soil Swab in puddle
0/1
1/1
0/1
1/1
1/1
1/1
1/1
0/2 0/2
1/1
1/1
1/1 0/1 1/4
Darkling beetles Swab on rodent carcass Rodents droppings Wild birds droppings
0/1 0/1 0/1
0/1 0/1
1/1
0/1
0/1
0/1
0/1
0/1
1/1 0/1 0/1
1/1
1/1
0/1
1/1
0/1 0/1
0/1
0/1 0/1 0/1
1/1
1/1
1/2
1/1
1/1 0/1
13
14
15
16
17
P
E
P
P
P
E
0/1 0/2 0/1 0/1
1/1 0/1 0/1 0/1
0/1
b
1/1
0/1 1/1 0/1
1/1
1/1
0/1 2/2
0/2
0/2
0/1
0/1
1/1 1/2
Water pipeline Water tank Well water
0/1
0/1
a
12
1/1
0/1 1/1
1/1
0/1 1/1
0/1
0/2
0/2
1/2
0/2
1/2
0/2
0/2 2/3 1/1 3/4
0/1 0/1
1/1
0/1
0/1 0/1
1/1
1/1
0/1 1/2
1/1 1/1 0/1
P: poultry; E: empty; aSlatted floors. bC.botulinum type D–C.
5/9
0/8
4/7
6/9
6/8
6/9
5/15
6/9
10/18
2/17
2/16
6/15
2/8 5/8
7/16
3/11 1/4
4/15
2/2
1/2
12/22 0/3 2/10
14/35
1/8 0/11 0/6
1/25
11/19 2/6 3/4
16/29
9/11 1/1 0/1 0/1
10/14
0/1 1/1 0/1
0/1 0/1 0/2
1/1
3/10
21/43
0/2 0/2
1/1
0/1 2/5
9/16 6/11 0/4 6/12
0/1 0/1
0/1 0/1 1/1
Total positive samples
7/11
1/2
2/17
73/185
C. botulinum in poultry farms in France 461
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House
1
R. Souillard et al.
Number of farms
462
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
Negave Posive
*
**
Poultry house
*
**
*
Changing room
**
*
Venlaon
**
*
Surroundings
**
*
Water
*
Feed Darkling beetles
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Figure 1. Detection of C. botulinum by real-time PCR in 17 affected poultry farms. *In the presence of poultry or shortly after their departure. **Post cleaning and disinfection.
Table 3. C. botulinum type C–D detection in each affected farm post cleaning and disinfection.
Farm number 8 House
Swab on the soil Soil Swab on the walls
9 0/2
0/2
0/1
0/1
0/2
0/1
Water
Water pipeline
Changing room
Swab on the walls
0/1
Ventilation
Swab in air admission Swab in air exit
1/1
Swab on the soil Soil Swab in puddle
1/1
Surroundings
Total a
10
a
11 0/1
a
12
13
15
16
0/1 0/1 0/1
0/1 0/1 0/1
0/2 0/2 0/2
1/1 0/2 0/1
0/2
0/2
0/1
0/1
0/1
0/1 0/1 1/1 0/1
0/1
0/1 0/1
0/2
0/1 0/1 0/1
0/1 0/2
1/11 0/6 0/9
1/26
0/5
0/5
0/1
0/5
0/5
1/1 2/2
0/1
3/8 2/3
5/11
0/1 1/2
0/1 0/2
1/8 1/7 0/1
2/16
0/1 2/5
1/6
0/10
0/3
0/6
0/10
Total positive samples
4/15
1/9
8/63
Slatted floors.
detection of low concentrations of C. botulinum (Vidal et al., 2011). This also ensures that only viable bacteria are detected, as dead bacteria or residual DNA are diluted in the enrichment broth, thus reducing the probability of detecting them during the subsequent PCR assay. Such an approach has recently been successfully used to develop a mostprobable-number PCR (MPN PCR) method to detect type E C. botulinum in samples collected from beaches on Lakes Michigan and Ontario and to demonstrate the presence of vegetative cells and not spore cells. In this study, all heattreated samples were negative for the bont/E gene whereas they were positive without heat treatment after enrichment, demonstrating that only viable cells were detected by PCR despite the presence of a small number of dead cells in the enrichment (Chun et al., 2013). In our study, to explore a large number of areas, we tested 21 different types of samples as appropriate for the specific circumstances of each farm, which explains the variability of sampling plan in each farm (from two to 18 samples) and the relatively low number of samples in each area (from 14 to 43 samples). The presence of C. botulinum type C–D in the environment of affected farms was detected in 39.5% of
the samples. C. botulinum was also detected after cleaning and disinfection of the poultry houses, indicating the difficulties of decontamination operations. This is illustrated by the number of recurrences among the investigated flocks and has been reported in previous studies (Okamoto et al., 1999). C. botulinum is considered ubiquitous. However, recent studies conducted in Norway and France in 46 farms without previously reported outbreaks (Hardy & Kaldhusdal, 2013; Souillard et al., 2013) showed that type C–D C. botulinum is not frequent in healthy poultry farms (only three positive samples in one out of the 46 farms investigated). This therefore indicates that positive samples detected in our study do not result from the normal presence of type C–D C. botulinum but are linked to botulism outbreaks. C. botulinum type C–D was detected in all positive samples, except in one swab in a changing room that was positive for type D–C. C. botulinum type C–D is clearly the most prevalent BoNT type associated with avian botulism at present in Europe (Woudstra et al., 2012). A study including different European countries showed that avian botulism is mostly associated with type C–D whereas
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C. botulinum in poultry farms in France 463
bovine botulism is associated with type D–C. However, in a few cases both types (C–D and D–C) were detected in turkeys, pheasants and wild ducks (Woudstra et al., 2012). Consequently, the type D–C detected in the changing room could be related to the botulism event in the poultry house. It is unknown whether the mosaic types are emerging or whether they are now being diagnosed because of the development of new methods that allow the discrimination of mosaic from non-mosaic types. In our study, C. botulinum type C–D was frequently detected in the litter. Litter has often been reported as being contaminated during botulism outbreaks. In a similar study, C. botulinum type C was detected in 26 out of 38 litter samples collected in affected broiler farms (Dohms et al., 1982). In another broiler outbreak, soil and litter were also reported as contaminated (Okamoto et al., 1999). Litter inevitably becomes contaminated with droppings and poultry carcasses. Dust essentially composed of contaminated droppings was also found to be positive with swabs from the walls of the houses. Spores of C. botulinum may be transferred from the affected houses to several areas. Indeed, we detected C. botulinum in the changing rooms, in the ventilation system and in the surroundings of the houses. C. botulinum may have been transported into the changing room via contaminated shoes or equipment. In the ventilation system, C. botulinum may have been disseminated by dust, which was found to be contaminated when sampled with swabs. Cleaning and disinfection of an air admission and exit circuit is not easy, because of the difficulty of accessing the system; this could explain the persistence of the spores after these operations. The surroundings of the houses were also frequently contaminated probably via farming activities (movement of the staff and the farmer, use of farm equipment) or via dust from the air exit system. Spores of C. botulinum are soil-associated and resistant in the environment. It has also been reported previously that mud or soil was contaminated by C. botulinum type C during outbreaks in wild birds (Franciosa et al., 1996) and in affected broiler farms (Okamoto et al., 1999). In our study, C. botulinum was found to have persisted in the soil inside the house and in the surroundings after decontamination operations. It appears to be difficult to ensure the complete destruction of spore forms in soil (Dohms, 2008), so this is an important factor to be addressed to prevent recurrence of the disease. Concerning animal reservoirs, invertebrates, notably fly maggots, are well-known vehicles of BoNTs during outbreaks of water bird botulism (Hubálek & Halouzka, 1991). C. botulinum type C was also recently observed in adult Calliphoridae and Sarcophagida flies collected around bird carcasses during a botulism outbreak (Vidal et al., 2013). In our study, darkling beetles were also found to be frequently positive. This suggests that darkling beetles could be vectors of C. botulinum and a reservoir of the bacteria. These beetles feed on bird droppings, bird carcasses or components of the litter, which could explain why these invertebrates have been shown to harbour C. botulinum (Hinkle & Hickle, 2008). Poultry eat darkling beetles, a natural source of food, and can thus become infected. Darkling beetles have already been shown to harbour several avian pathogens such as Escherichia coli, Salmonella spp. and Campylobacter spp. (Hinkle & Hickle, 2008). Beetles can actually be involved in Salmonella enterica transmission between two consecutive broiler flocks (Skov et al., 2004) and S. enterica can transit in the gut of darkling beetles, inducing a dispersion of viable pathogenic bacteria
within 2 to 3 h (Zheng et al., 2012). Darkling beetles are abundant in poultry production and difficult to suppress. Disinfestation is thus crucial in handling botulism outbreaks and preventing recurrence. Rodents are also a reservoir of a variety of pathogens (Hinkle & Hickle, 2008) and notably of C. botulinum (Popoff, 1995). Here one swab collected from a rodent carcass was found to be positive. Whether the rodent carcass was the source of the outbreak or the result of cross-contamination was not determined. In a turkey botulism outbreak, C. botulinum was also isolated from the liver of a dead rat found close to the barn (Popp et al., 2012). This illustrates how important the management of rodents is to prevent dissemination of the disease. Water, frequently found to be positive in our study, has already been suspected as a source of contamination during a bird outbreak in a zoo (Raymundo et al., 2012) and C. botulinum has been detected in mud samples obtained from different raw-water storage areas that were affected by waterfowl botulism in the Netherlands (Notermans et al., 1980), but few data are available concerning water contamination in poultry farms. In a recent case report, no water sample, taken from pipes and Plasson (bell) drinkers, was positive (Popp et al., 2012). In our study, water samples from the distribution system may have been contaminated by dust in the tank or by biofilm in the pipelines. The hydrophobicity of C. botulinum spores could enable them to adhere to surfaces and biofilm formation (Wiencek et al., 1990). Moreover, two well-water samples were positive. This well water is used as drinking water and could also explain the contamination of water pipeline samples. The well water could be contaminated by spores of C. botulinum, with carcasses of rodents or other small animals. Further investigations are needed to better understand the role of drinking water as a source of contamination in poultry farms. Finally, only one feed plate was positive, probably contaminated by droppings or dust. It is difficult to detect C. botulinum in feed, probably because of the sampling method. Few reports have mentioned it, but feed could be a source of contamination (Dohms et al., 1982). This study identifies the critical contaminated areas in poultry farms after a botulism outbreak. Darkling beetles, drinking water, the ventilation system and soil from the surroundings and the houses are the main contaminated areas that have to be taken into account for handling botulism outbreaks. These findings are essential for implementing control of the disease and will enable us to assess a decontamination procedure to prevent the recurrence of avian botulism.
Acknowledgements The authors are grateful to the poultry farmers, veterinarians and technicians. They would like to thank Denis Léon, Sandra Rouxel, Emmanuelle Houard and Valéria Guimaraes for their participation in the study.
Funding This project was made possible by financial support from the French Ministry of Agriculture and the French Agency for Food, Environmental and Occupational Health & Safety.
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