PROCEEDINGS PROCEEDINGS

O26 Material and Methods Our study was carried out on three on-farm biogas Reduction of sporulating and non-sporulating plants (BGP1, BGP2 and BGP3), two filled with pig pathogens during anaerobic digestion of manure (BGP1 and BGP3) and one with bovine manure livestock manure in biogas plants (BGP2). Over one-year, they were visited eight Denis M.1, Druilhe C.2, Le Maréchal C.1, Repérant times each. At each visit, three replicates of E.1, Boscher E.1, Nagard B.1, Rouxel S.1, both inputs (manure) and digestates were collected Poezevara T.1, Martin L.1, Pourcher A.-M.2 for detection and enumeration (MPN/g) of Salmonella Hoa Binh 1Anses - Laboratoire de Ploufragan, Ploufragan, France, spp, Listeria monocytogenes, Campylobacter sp p., 2IRSTEA, Rennes, France Clostridioides difficile and Clostridium botulinum. A total of 144 samples (72 inputs, 72 digestates) Introduction were analyzed. In the current context of developing renewable energies and recovering organic waste, on-farm Results anaerobic digestion (AD) represents a major challenge All the pathogens were detected in manure at a Figure 1: Sampled communes in Da Bac district of Hoa Binh province for the agricultural sector (energy and organic frequency of 33.3 % (C. botulinum), 8 8 % (C. difficile), recovery of livestock manure and agricultural 92 % (Campylobacter spp.), and 95.8 % (Salmonella and substrates). In France, most of biogas plants fed with Listeria monocytogenes) and in all three BGP, except

manure operate at mesophilic conditions converting C. botulinum which was not detected in manures of Oral Presentations References organic matter to biogas and by-product degradation, BGP1 and BGP2. i.e. digestate. This digestate is usually spread as The pathogens were also detected in digestate at (1) Van, D.N., Thi, N., V, Dorny, P., Vu, T.N., Ngoc, fertilizer on land after transformation or storage. a frequency of 37.5 % (Campylobacter sp p.), 79.2 % M.P., Trung, D.D., Pozio, E., 2015. Trichinellosis in Farm animals like pig, bovine and poultry are known to (C. botulinum), 8 3.3 % (L. monocytogenes), 87.5 % Vietnam. Am. J. Trop. Med. Hyg. 92, 1265-1270. be reservoirs of various pathogenic microorganisms (Salmonella spp.) and 100 % (C. difficile). However, no (2) Unger F, Chau Thi Minh Long and Nguyen Viet responsible of animal or human infections (Denis et Campylobacter spp. could be isolated from digestates Khong. Prevalence of trichinellosis and cysticer- al., 2011; Boscher et al., 2012, Souillard et al., 2014 of BGP2. cosis in indigenous pigs from ethnic minorities and 2015, Moono et al., 2016; Gosling et al., 2018; In manure, the level in MPN/g varied in mean from for selected communes in the Central Highlands Thépault et al., 2018). Because these pathogens can 249 to 368 for Campylobacter, from 1.1 to 359.1 for (Dak Lak). Nairobi, Kenya: ILRI; 2016. survive in manure, their fate during mesophilic AD Salmonella, from 3.1 to 145.9 for L. monocytogenes, appears to be a matter of public health concern. from 0.5 to 234.5 for C. difficile and from 0 to 3.5 In this study, we investigated the effect of for C. botulinum (Fig. 1). mesophilic AD on the level of sporulating pathogens (Clostridioides difficile and Clostridium botulinum) and non-sporulating pathogens (Salmonella spp, Listeria monocytogenes and Campylobacter sp p.).

Campylobacter Salmonella L. monocytogenes C. difficile C. botulinum 800 800 800 800 800

600 600 600 600 600

MPN / g 400 400 400 400 400 +

200 200 200 200 200

+ 0 + 0 + 0 0 0 man. dig. man. dig. man. dig. man. dig. man. dig. + mean value median value

Figure 1: Concentrations of the pathogens in manures and digestates

102 | SafePork 2019 Figure 1. Concentrations of the pathogens in manures and digestates SafePork 2019 | 103 PROCEEDINGS PROCEEDINGS

In raw digestate, the level in MPN/g varied in mean Denis M, Henrique E, Chidaine B, Tircot A, et al. O27 Materials and Methods from 0 to 6.3 for Campylobacter, from 1.1 to 6.9 for (2011) Campylobacter from sows in farrow-to-finish In 2018, a total of 250 fattening pigs from 25 farms Salmonella, from 3 to 45.7 for L. monocytogenes, from pig farms : risk indicators and genetic diversity. Hepatitis E - analyzing the occurrence in (10 pigs per farm) were sampled in an abattoir in 8.2 to 80.1 for C. difficile and from 0.3 to 2.4 for Veterinary Microbiology 154: 163-170. slaughter pigs for a risk assessment of raw North-West Germany. One sample of ham muscle, one C. botulinum (Fig. 1). Concentration of C. botulinum Froschle, B., Messelhausser, U., Holler, C., Lebuhn, M. meat products sample of liver tissue and one sample of the muscle was therefore very low in both samples, manure and (2015). Fate of Clostridium botulinum and incidence Dzierzon J.1, Oswaldi V.1, Merle R.2, of the diaphragm pillar were collected from each pig raw digestate, with a maximum of 13 MPN/g. of pathogenic clostridia in biogas processes. J Appl Langkabel N.1, Meemken D.1 during the slaughter process. Each animal was tagged During AD, the average level of pathogens decreased Microbiol, 119, 936-47. 1Freie Universität Berlin, Institute of Food Safety and Food individually and samples were taken at different 2 between manure and digestate by 2 Log10 (Salmonella Gosling RJ, Mueller-Doblies D, Martelli F, Nunez- Hygiene, Berlin, Germany, Freie Universität Berlin, Institute stages of the slaughter line. Livers were collected spp.), 0.3 Log10 (L. monocytogenes), 2.1 Log10 Garcia J, et al., (2018) Observations on the for Veterinary Epidemiology and Biostatistics, Berlin, Germany and stored in boxes during the slaughter process as

(Campylobacter spp.), 0.4 Log10 (C. difficile) and 0.1 distribution and persistence of monophasic Salmonella usual until sampling. All samples were chilled and

Log10 (C. botulinum). Typhim uriu m on infected pig and cattle farms. Vet Introduction transported to the institute’s laboratory. Muscle Microbiol.227:90-96. The hepatitis E virus (HEV) of genotype 3 and 4 is samples from the diaphragm pillar were stored at Discussion and Conclusion Kearney, T. E., Larkin, M. J., Frost, J. P., Levett, known as a zoonotic agent. In this context, the -30 °C and liver and ham muscle samples were stored Our study showed that non-sporulating pathogens like P. N. (1993). Survival of pathogenic bacteria during pig was identified as the main animal reservoir. at -80 °C until laboratory examination. Salmonella spp, Listeria monocytogenes, Campylobacter mesophilic anaerobic digestion of animal waste. J In Europe, the consumption of raw or undercooked To determine the seroprevalence, meat juice from the spp. can be detected in digestate after anaerobic Appl Bacteriol, 75, 215-9. pork products represent a potential risk for HEV diaphragm pillar samples was serologically tested for digestion like in previous studies (Kearney et Moono P, Foster NF, Hampson DJ, Knight DR, et al. infections in humans. In humans, HEV infections can HEV antibodies using the PriocheckTM HEV Antibody

al., 1993; Bonetta et al., 2011; Orzi et al., 2015) (2016) Clostridium difficile infection in production cause acute hepatitis, which is usually self-limiting. porcine ELISA Kit (Thermo Fisher Scientific®, USA) Oral Presentations suggesting that these pathogens can survive this animals and avian species: a review. Foodborne Pathog Chronicity in immunocompromised patients and a high according to the manufacturer’s manual. The liver process, even if their concentrations are reduced Dis. 2016 Dec;13(12):647-655. mortality rate of up to 28 % in pregnant women have and muscle samples were analysed for the presence of during the process. C. botulinum concentration was Orzi, V., Scaglia, B., Lonati, S., Riva, C., et al. been reported (Meng 2011). HEV RNA by real-time RT-PCR according to Jothikumar very low, whether in manures or in digestates, which (2015). The role of biological processes in reducing In Germany, according to § 7 of the German Infection et al. (2006) after RNA extraction with the RNeasy® confirms study of Froschle et al, (2015). In this both odor impact and pathogen content during Protection Act (IfsG, 2019), the direct or indirect Mini QIAcube Kit (QIAGEN®, Germany). study, C. difficile was also frequently detected mesophilic anaerobic digestion. Sci Total Environ, detection of HEV in humans must be reported to For each pig the antibody status will be gathered in digestate with similar levels of C. difficile 526, 116-26. official health services. In 2018, a total of 3,275 and herd status will be analysed, too. Afterwards, concentration. Souillard R, Woudstra C, Le Maréchal C, Dia M, et cases of hepatitis E was reported to the Robert the presence of HEV antibodies for each animal will With this one-year survey, we demonstrated that al. (2014) Investigation of Clostridium botulinum in Koch Institute (RKI 2019). be compared with the presence of viral RNA in the mesophilic AD does not lead to bacterial growth commercial poultry farms in France between 2011 and As pigs are a main reservoir of HEV several studies liver and the muscle. and even reduced concentration of sporulating and 2013. Avian Pathol. 2014;43(5):458-64. were performed identifying the antibody status of non-sporulating pathogens. Thus, such treatment of Souillard R, Le Maréchal C, Hollebecque F., Rouxel S. fattening pigs across EU member states. With a Results livestock manure can be effective in reducing the et al., (2015) Occurrence of C. botulinum in healthy seroprevalence of up to 96 % (Wutz et al. 2013), HEV In total, 62 % (155/250) of the meat juice samples presence of these pathogens, and reduce consequent cattle and their environment following poultry shows a wide distribution among fattening pigs in were positive for antibodies against HEV at a single spreading in the environment after post-treatment botulism outbreaks in mixed farms. Vet Microbiol Germany. Nevertheless, national studies examining animal basis. At herd level, 72 % (18/25) of the herds (eg. storage or post-digestion) of digestates. Volume 180: 142-145. the occurrence of HEV RNA in liver or muscle samples were positive. Herds were considered to be positive, Thépault A, Poezevara T, Quesne S, Rose V, et al. from pigs are rare. if at least one of the ten samples was positive. Acknowledgements (2018) Prevalence of thermophilic Campylobacter The objective of this study was to estimate the risk For the herd seroprevalence four groups, according The authors are grateful to the participating in cattle production at slaughterhouse level in of HEV entering the food chain via pork products to the serological detection rate, were defined. The farmers. This research was financially supported France and link between C. jejuni bovine strains based on serological tests and on the analysis of herds investigated were allocated to one of these by the French Environment and Energy Management and campylobacteriosis. Front Microbiol. 2018 Mar pork liver and muscle samples from the same animal groups using their antibody prevalence (Table 1). Agency (ADEME). 19;9:471. used for the production of pork liver and pork meat products. References Table 1: Allocation of herds according to the antibody status Bonetta, S., Ferretti, E., Bonetta, S., Fezia, G., serological detection rate Proportion of herds (n/N) Carraro, E. 2011. Microbiological contamination of digested products from anaerobic co-digestion of 0 % (HEV seronegative) 28 % (7/25) bovine manure and agricultural by-products. Lett 10 %-30 % (low prevalence) 8 % (2/25) Appl Microbiol, 53, 552-7. Boscher E., Houard E., Denis M. (2012) Prevalence 60 %-90 % (high to very high prevalence) 16 % (4/25) and distribution of Listeria monocytogenes serotypes 100 % (all samples are HEV seropositive) 48 % (12/25) and pulsotypes in sows and fattening pigs in farrow- to-finish farms (France, 2008). Journal of Food Protection 75 (5):889-895

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