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.
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Clostridium botulinum is an anaerobic spore-forming bacterium that produces botulinum neurotoxins (BoNTs) causing botulism, a neuroparalytic disease affecting both humans and animals. Among the seven BoNTs (from A to G) that have been characterised to date, human botulism is mainly associated with types A, B, E and F,1 while animal botulism is more frequently caused by C/D, D/C, C and D.1–3 Animal botulism affects mainly poultry and cattle.4 The disease induces flaccid paralysis and high mortality in affected farms ranging between 2.8per cent and 35per cent in 17 investigated poultry outbreaks with significant economic losses.5 Paralytic signs progress cranially from the legs to include wings, necks and eyelids, and death results from cardiac and respiratory failure.1 Outbreaks have been reported worldwide. Animal botulism is not a notifiable disease in most countries, which results in the absence of official data to evaluate the extent of the disease. Botulism is nevertheless considered as an emerging problem in Europe.6 In France between 2013 and 2019, 153 outbreaks were reported in various avian species.7 In Italy, from 2007 to 2018, 40 outbreaks in poultry were diagnosed affecting either broilers, ducks, turkeys, laying hens or goose.8 Many poultry productions in free ranged or enclosed rearing conditions can be affected by botulism.9–12
It has been demonstrated that litter is often contaminated after an outbreak of poultry botulism.5 13 Moreover, C botulinum spores are known to be able to persist for years in the environment.14 15 Surprisingly, despite this context, few studies have been conducted up to now to evaluate the contamination of poultry manure after an outbreak,16 the outcome of the bacteria over time or measures that should be implemented to handle contaminated manure. These points are of major importance, however, given that botulism frequently recurs in affected poultry farms5 13 17 and that cases of cross-contamination between farms and animal productions—especially between poultry and cattle—due to contaminated manure are often reported.18–20
Although notification of botulism is compulsory in France for all animal species,21 there is no specific national decree at the moment specifying the management measures of animal botulism and particularly manure removal. According to the French and European regulations of animal by-products,22 23 manure can be spread over land if the competent authority considers that there is no risk of contamination. In the event of any notifiable animal disease, when transporting manure to the nearest burning plant increases the danger of microbial dissemination, the competent authority may, by derogation, authorise its disposal by burning or burial on site.
Recommendations regarding the specific management of manure on farms affected by botulism have been put forward by the French Food Safety Agency (AFSSA) and the Federal Agency for the Safety of the Food Chain (AFSCA).24 25 The composting or landfill of manure can create anaerobic and low-temperature conditions favourable to the growth or survival of C botulinum.24 Consequently, according to the AFSSA report, the only economically viable solution for dealing with manure after a botulism outbreak is its incineration under firefighter control. In Belgium, AFSCA has reported that the spreading of manure contaminated by C botulinum following a bovine botulism outbreak can potentially increase spore concentration in the environment, leading to a higher direct risk of intoxication for cattle.25 Although this risk was assessed as ‘very low’, the AFSCA Scientific Committee has recommended specific measures to minimise environmental contamination like incineration of the manure, treatment with quicklime and spreading on crops only, not on grazed or silage grassland, using an injection procedure to avoid spore dissemination.
The manure management in poultry farms affected by botulism involves potential health hazard from an environmental, public and animal health point of view. Despite the risk of dissemination of C botulinum in the environment that may lead to recurrence of the disease or cross-contamination between productions, very few data are available on the manure contamination and its management in a context of botulism outbreak. The aims of this study were to1 describe the management of poultry manure on 10 poultry farms affected by botulism and to2 assess the contamination and persistence of C botulinum in manure over time.
Materials and methods
Manure sampling and epidemiological data collection on poultry farms
From 2015 to 2017, 10 poultry farms affected by botulism were investigated. On a voluntary basis, avian veterinarians aware of the study reported the outbreaks and farm selection was based on the farmers agreeing to take part in the study. Two visits were conducted on each farm after manure had been removed from the poultry house and stored outside: the first visit was planned two weeks after manure removal and the second one two months later. During each visit, 15 samples were collected in the manure pile: five samples (two on the surface and three deep down) were taken from three different parts of the pile. Each manure sample was collected in a 200 ml container. The deep samples were extracted at a depth of 50 cm. The manure temperature was also measured during sampling using a probe. The sampling protocol of manure was based on previous epidemiological investigations in which environmental samples were collected (soil, manure or silage) to detect C botulinum.5 20 A questionnaire was also completed with the farmers to collect epidemiological data on farming practices and poultry health: description of the poultry house, the flock, the outbreak of botulism (age and sexes of animals affected, mortality and botulism BotNT type) and the management of the manure after the departure of poultry with the operations in the house and after being moved outside.
Analysis of manure samples
Whole samples of manure were half diluted (manure was weighted and an equivalent volume of broth was added) in prereduced trypticase-peptone-glucose-yeast extract broth (TPGY) then manually homogenised. Fifty grams of this diluted solution was diluted five times (addition of 200 ml of TPGY) and finally homogenised using a Pulsifier (Microgen Bioproducts, Camberley, UK) for 15 seconds, according to the recommendations of Standard NF EN ISO 6887–6.26
Incubation was then performed for at least four days at 37°C under anaerobic conditions (Station A35, Don Whitley, distributed by Biomerieux, Bruz, France). They were then stored several months at a temperature below −18°C before DNA extraction. The DNA was extracted from 1 ml of each enriched extract using the NucleoSpin Soil kit (Macherey-Nagel, Duren, Germany). Genes encoding BoNT type C, D, C/D and D/C were detected using real-time PCR as previously described.16
Epidemiological data collected on the 10 poultry farms affected by botulism were described: the poultry flock, the outbreak of botulism and the management of the manure. The influence of specific characteristics of manure (temperature and sampling in surface or depth) on the detection of C botulinum was studied using chi-squared test and Wilcoxon rank-sum statistical test, considering a p value <0.05 to be statistically significant.
Management of poultry manure after a botulism outbreak on 10 poultry farms
A description of the botulism outbreaks investigated here is detailed in table 1. Different poultry productions and BoNT types were included in this study: six turkey farms (three of type D/C, one of type D and two of type C/D), three broiler farms (of type C/D) and one laying-hens farm (of type C/D). The 10 farms were investigated between 2015 and 2017 and the outbreaks occurred systematically between March and November. Whatever the poultry species, the mortality rate ranged between 2per cent and 49per cent. It is noteworthy that most outbreaks affected males, most of the time after the departure of females for slaughtering.
Manure management is presented in table 2. For each farm, samples of manure were collected from a single pile, except for farm no. 4 for which two piles of manure were sampled, because manure was removed at two times due to the different departure times of females and males. The way manure is handled varies greatly from one farm to another. Quicklime was incorporated into five piles of manure: both inside the house and outside it during the manure stockpiling for one farm, only in the house for one farm and only outside the house during manure stockpiling for three farms. It is noteworthy that the use of quicklime requires the compliance with the legislation and safety measures (protection of the persons and risk of fire, eg, of the house when inside use). Combustion was noticed in three out of the five manure piles treated with quicklime, so during the visits, combustion was in progress and only unburnt manure residues remained. None of these three piles were covered with a tarpaulin. The other two piles treated with quicklime without signs of combustion were covered with a tarpaulin and had not evolved between the two visits. On the other farms, four piles of manure were just stored outside (including one covered with a tarpaulin), one pile was stored outside but bacterial flora had been added (through a commercial additive combining lactic acid bacteria and Bacillus) before it was covered with a tarpaulin and one pile of manure was incinerated on the site (according to the current legislation). The manure, including the residual unburnt manure, was spread over the farmland using an injection process, whatever the prior treatment.
Clostridium botulinum detection in manure samples
The results of C botulinum detection in poultry manure are presented in table 3. C botulinum was detected in 161/285 (56.5per cent) of investigated manure samples. C botulinum was detected significantly more frequently in samples collected during visit 2 (on average 64 days after the removal of manure from the poultry house) than during visit 1 (on average 12 days after its removal) (65.8per cent vs 49.7per cent, P<0.01). Regarding manure temperature, C botulinum was detected significantly more frequently at lower temperatures, that is, the mean temperature was 38.7°C for negative samples and 28.1°C for positive samples (P<0.01). C botulinum tended to be found more frequently on the surface of the pile than deeper down (63.1per cent vs 50per cent, P=0.025). The average temperature for samples collected on the surface was 21.1°C, while it was 44.2°C for samples collected deeper down.
By adding quicklime to the manure, three out of five manure piles were subject to combustion, thus reducing their volume. Quicklime had no effect on the detection of C botulinum in either the manure or in its unburnt residues in the event of combustion: C botulinum was detected in 53per cent (72/135) of the manure samples to which quicklime had been added and in 59per cent (89/150) of the manure samples without any addition of quicklime. The bacterial flora added to one pile of manure did not have an effect on the detection of C botulinum over time either.
To the authors’ knowledge, this study is the first to demonstrate the systematic and durable contamination by C botulinum of poultry manure after a botulism outbreak and the role of temperature in the detectability of this pathogen. The survival of pathogens in manure varies markedly depending on the microorganism’s characteristics, the physical properties of the manure and the climatic conditions.27
This study shows that various methods (classical storage, addition of quicklime, bacterial flora or incineration) are implemented to deal with manure following an outbreak of poultry botulism. Botulism is a notifiable disease in France,21 so it is compulsory to report it to government authorities to ensure that the disease is controlled in accordance with French legislation, taking into account health, safety and environmental requirements. However, there is no specific national decree at the moment to describe the measures that should be implemented to manage animal botulism, which may explain the diversity of manure practices on farms.
In the 10 poultry farms affected by botulism, positive samples accounted for 56.5per cent of all the manure samples. C botulinum was still detected in the residual unburnt manure of the three piles subject to combustion with quicklime. The remaining residues appear to be as contaminated as the manure without any quicklime. The number of positive samples was higher after on average 64 days of manure storage (65.8per cent) than during the initial sampling on average 12 days after the manure had been removed from the poultry house (49.7per cent). Two hypotheses could explain the observed increase in the number of positive manure samples over time. First, this could be due to the growth of C botulinum in manure, a potentially favourable medium for the bacterium with anaerobic conditions and the presence of organic material. The other hypothesis is that there is a decrease in bacterial species able to inhibit C botulinum growth, allowing the pathogen to be detected more easily in the samples collected during the second visit than in the initial ones.28 29 Further investigation is required to explain this increase.
Regarding conditions associated with C botulinum detection in poultry manure, this study shows that C botulinum was detected significantly more frequently at lower temperatures (28.1°C vs 38.7°C) and more frequently on the surface, with a mean temperature of 21.1°C vs 44.2°C deeper in the pile. The optimal growth temperature for C botulinum is between 30°C and 42°C, although it can survive between 10°C and 48°C30 depending on the BoNT types and C botulinum group. The mean temperature measured at a depth of around 50 cm is most frequently within this interval and could allow the initiation of spore germination. The environmental conditions present (pH and bacteria flora among others) could induce an inactivation of vegetative cells. This phenomenon has already been reported for Clostridium difficile, whose indirect inactivation at 55°C in sewage sludge was demonstrated via spore germination and inactivation of vegetative cells.31 Nevertheless, further investigation is required in order to identify the factors explaining these results.
Only a small number of farms was investigated in this study, this limit is to be considered when interpreting the results. The 10 botulism outbreaks were reported by veterinarians in commercial poultry farms, this explain the various poultry productions affected with layers, broilers and turkeys. The farm selection was also based on the farmers agreeing to take part in the study. The sampling protocol of the manure pile was based on the previous sampling methodologies described in the environment to detect C botulinum.5 20 A representative sampling of a pile of manure is difficult to obtain, given the heterogeneity of the manure and its various physical properties influencing the growth of pathogens. Considering these characteristics and the field logistical and laboratory constraints, 15 samples were taken in three parts of the pile both on surface and in depth with temperature measurement.
Despite the limitations of the sampling design, this study highlights the durable persistence of C botulinum in poultry manure after a botulism outbreak in field conditions. Indeed, the storage of manure over several months did not allow its elimination. The benefit of using quicklime (according to the legislation and safety measures) lies in the subsequent combustion of the manure and a reduction in volume, but the unburnt manure remained contaminated. Consequently, whatever the management of the manure—whether using conventional storage or adding quicklime—the manure, or its residues in the event of combustion, is still contaminated by C botulinum and must be carefully managed to avoid cross-contamination between productions and recurrence of the disease on the same farm. Spreading manure contaminated by C botulinum on farmland implies the probable contamination of soil. It has already been demonstrated that spreading compost spiked with C botulinum spores over soil induces a persistent contamination of the soil with deep dissemination after spreading.32 Another study has demonstrated under laboratory conditions the systemic colonisation of clover (Trifolium repens) by C botulinum when the clover is grown on contaminated soil.33 Considering these results, it may be assumed that spreading manure contaminated by C botulinum over land may be risky from an environmental, public and animal health point of view and may also lead to dissemination of the germ during this operation via the material and vehicles.
This study shows that various manure management procedures are implemented on farms after an outbreak of poultry botulism, and that these strategies do not appear satisfactory because they do not reliably destroy C botulinum spores. Whatever the BoNT type involved in an outbreak of botulism, poultry manure is contaminated by C botulinum, and this contamination persists over time. Given the potential health hazard this represents, it is crucial to implement appropriate measures for managing manure after an outbreak to avoid cross-contamination and prevent recurrence of the disease. Further studies are required to provide efficient solutions that are both relevant and applicable on the field for properly handling manure after an outbreak of botulism so as to avoid residual contamination and spore dissemination.
The authors would like to thank the farmers and veterinarians for their participation in the study.
Funding This study was financially supported by the ANIBOTNET project (ANIHWA call), the French Ministry of Agriculture, the French Agency for Food, Environmental and Occupational Health Safety and the Côtes d’Armor departmental council.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. Additional data are available on request.
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