Quantification of indicator and pathogenic bacteria in manures and digestates from three agricultural biogas plants over a one-year period.

Interest in the conversion of manure in biogas via anaerobic digestion (AD) is growing, but questions remain about the biosafety of digestates. For a period of one year, we monitored the impact of three mesophilic agricultural biogas plants (BPs) mainly fed with pig manure (BP1, BP3) or bovine manure (BP2) on the physicochemical parameters, the composition of the microbial community and the concentration of bacteria (E. coli, enterococci, Salmonella, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum and Clostridioides difficile). The BP2 digestate differed from those of the two other BPs with a higher nitrogen content, more total solids and greater abundance of Clostridia MBA03 and Disgonomonadacea. Persistence during digestion ranked from least to most, was: Campylobacter (1.6 to >2.9 log10 reduction, according to the BP) < E. coli (1.8 to 2.2 log10) < Salmonella (1.1 to 1.4 log10) < enterococci (0.2 to 1.2 log10) and C. perfringens (0.2 to 1 log10) < L. monocytogenes (-1.2 to 1.6 log10) < C. difficile and C. botulinum (≤0.5 log10). No statistical link was found between the reduction in the concentration of the targeted bacteria and the physicochemical and operational parameters likely to have an effect (NH3, volatile fatty acids and total solids contents, hydraulic retention time, presence of co-substrates), underlining the fact that the fate of the bacteria during mesophilic digestion depends on many interacting factors. The reduction in concentrations varied significantly over the sampling period, underlining the need for longitudinal studies to estimate the impact of AD on pathogenic microorganisms.

Controlled Experimental Infection in Pigs with a Strain of Yersinia enterocolitica Harboring Genetic Markers for Human Pathogenicity: Colonization and Stability.

Yersinia enterocolitica (Ye) is one of the major causes of foodborne zoonosis. The BT4/O:3 bioserotype is most commonly isolated in human infections. Pigs are considered the main reservoir of Ye, and hence, understanding the dynamics of infection by this pathogen at the individual and group levels is crucial. In the present study, an experimental model was validated in Large White pigs infected with a BT4/O:3 strain. This study showed that Ye contamination in pigs may occur via the introduction of the bacteria not only by mouth but also by snout, with a colonization process consisting of three periods corresponding to three contamination statuses of pigs: P1, corresponding to the 24 h following ingestion or inhalation of Ye with the appearance of bacteria in tonsils or in feces; P2, from 2 days postinoculation (dpi), corresponding to expansion of Ye and colonization of the digestive system and extraintestinal organs associated with an IgG serological response; and P3, after 21 dpi, corresponding to regression of colonization with intermittent Ye detection in tonsils and feces. Although the inoculated strain persisted up to 56 dpi in all pigs, genetic variations with the loss of the gene yadA (a gene involved in human infection) and the emergence of two new multilocus variable-number tandem-repeat analysis (MLVA) profiles were observed in 33% of the 30 isolates studied. This experimental infection model of pigs by Ye provides new insights into the colonization steps in pigs in terms of bacterial distribution over time and bacterial genetic stability.

Longitudinal study describing time to Salmonella seroconversion in piglets on three farrow-to-finish farms.

BACKGROUND: Pigs are frequently colonised with Salmonella enterica, and this constitutes a major risk for human salmonellosis. The infection can be assessed by the serological response of pigs to S enterica. A longitudinal study was undertaken on-farm to correctly describe this serological response and investigate factors associated with age at Salmonella seroconversion. METHODS: Three pig farms and in each farm three successive batches were considered. Per batch, 40 piglets were selected at random from 10 sows (four piglets per sow). Blood was sampled from sows one week after farrowing and from piglets at weeks 1, 6, 10, 14, 18 and 22 and at the slaughterhouse. Salmonella antibodies were detected in serum using a commercial ELISA test. Factors related to farm characteristics, batch management system, porcine reproductive and respiratory syndrome infection, and sows' Salmonella serological status were recorded to assess their effect on age at seroconversion. RESULTS: At week 1 after farrowing, 96.5 per cent of the sows had antibodies against Salmonella. The serological results of piglets at weeks 1 and 6 only were positively correlated with those of the sows. The average age at Salmonella seroconversion was 137±2.2 days (confidence interval at 95 per cent). The first seroconversions occurred from weeks 10 to 14, but most of the pigs (54.6 per cent) were seropositive at the end of the fattening period, with variations between farms and batches (28.9-75.7 per cent). Herd/farm was significantly associated with age at seroconversion. CONCLUSION: This longitudinal study allowed the authors to follow precisely the evolution of Salmonella seroconversion from maternity to slaughterhouse and confirm the relationship between the seroconversion of sows and serology of their piglets. Moreover, factors related to farm practices and management as a whole are more influential than individual factors (at the pig level) on age at Salmonella seroconversion.

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants.

The number of agricultural biogas plants has been increasing in the past decades in some European countries. Digestates obtained after anaerobic digestion (AD) of manure are usually spread on agricultural land; however, their hygiene status regarding pathogens posing public health and/or animal health challenges has been poorly characterized up to now in France. In this study, three replicates of manure and digestate were collected from five farm biogas plants receiving animal manure in order to assess the occurrence and concentrations of sporulating (Clostridium botulinum, Clostridioides difficile, Clostridium perfringens) and nonsporulating (Listeria monocytogenes, thermotolerant Campylobacter spp., Salmonella, Escherichia coli, enterococci) bacteria. Concentrations of E. coli, enterococci, and C. perfringens in digestates ranged from 102 to 104 , 104 to 105 , and <103 to 7 × 105  CFU/g, respectively. Salmonella and C. difficile were detected in manure and digestate from the five biogas plants at concentrations ranging from <1.3 to >7 × 102  MPN/g and from 1.3 to 3 × 102  MPN/g, respectively. Thermotolerant Campylobacter, detected in all the manures, was only found in two digestates at a concentration of cells ranging from <10 to 2.6 × 102  CFU/g. Listeria monocytogenes and C. botulinum were detected in three manures and four digestates. The bacterial counts of L. monocytogenes and C. botulinum did not exceed 3 × 102 and 14 MPN/g, respectively. C. botulinum type B was detected at very low level in both the manure and digestate of farm biogas plants with no botulism history. The levels of pathogenic bacteria in both manure and digestate suggested that some bacteria can persist throughout AD.

Experimental infection of pigs by Salmonella Derby, S. Typhimurium and monophasic variant of S. Typhimurium: Comparison of colonization and serology.

Salmonella serovars Derby, Typhimurium and the monophasic variant of Salmonella Typhimurium are the most frequently isolated serovars in pigs in France. To compare the excretion patterns, seroconversion to Salmonella and contamination of the organs of pigs inoculated with strains of all three serovars, we conducted an experimental trial with 28 SPF piglets. Four were used as a negative control, while the other 24 were divided equally into three groups. Each group was inoculated at 7 weeks of age with a different strain: S. Derby (SDb), S. Typhimurium (ST), and the monophasic variant of S. Typhimurium (mST). Fecal and blood samples were collected twice a week up until necropsy, on 21 days post-inoculation (DPI) for half of each group and 49 DPI for the remaining piglets. During necropsy, the tonsils, mesenteric lymph nodes and various intestinal contents were collected from each pig. Salmonella bacteria were quantified in CFU/g by a bacteriological method, and levels of Salmonella antibodies were measured using an ELISA Kit. Piglets inoculated with mST continuously excreted Salmonella in their feces throughout the trial. For each of the other serovars, one piglet was Salmonella-negative on one DPI. The quantity of Salmonella excreted was statistically different between the group inoculated with ST and mST (p < 0.05), but no differences were found between the other serovars. The tonsils, cecum and jejunum were the most contaminated organs in all groups. Seroconversion for all the piglets was completed by different DPI: 28 for ST, 31 for mST and 38 for SDb. No major differences were found in terms of excretion and colonization among the studied serovars.

Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium.

The monophasic variant of Salmonella Typhimurium is highly prevalent in human and in pork. However, little is known about colonization dynamics and serology in pigs. We orally inoculated 24 seven-week-old piglets with 109 CFU/pig of a porcine strain of monophasic Salmonella Typhimurium in an experimental trial. Three groups of eight piglets were orally inoculated and monitored for 21, 49, or 84 days post-inoculation until necropsied. From 3 days post-inoculation to necropsy, individual feces were sampled twice weekly and blood once weekly. At necropsy, the tonsils, mesenteric lymph nodes, and the contents of the duodenum, jejunum, ileum, and cecum were collected from each pig. We determined the number of CFU/g in all the samples and measured also Salmonella antibodies in OD% in all blood samples. At different times during the trial, we tested by MLVA (Multilocus Variable Number Tandem Repeat Analysis) the genomic stability of the strain after passing through the intestinal tract. Salmonella was continuously excreted by pigs, ranging from 1.4 to 5.8 log10 CFU/g. At necropsy, Salmonella was present in all samples, but the tonsils were particularly infected. Salmonella antibodies were detected in five pigs 7 days post-inoculation. At 49 days post-inoculation, all the pigs were seropositive. We observed new MLVA types for 3.3% of the isolates tested over the trial. Our study allowed us to show the serovar's ability to persist in pigs after infection up to 84 days post-inoculation. We demonstrated that Salmonella seroconversion appeared earlier than in naturally infected pigs and that the strain's genome can evolve after passing through the digestive tract of pigs.

Isolation of Salmonella enterica in laying-hen flocks and assessment of eggshell contamination in France.

The present investigation was conducted in conjunction with the European Union baseline study for the estimation of Salmonella prevalence in laying-hen flocks. It aimed at evaluating eggshell contamination in farms positive for Salmonella, characterizing the genetic patterns of Salmonella strains and identifying the factors associated with Salmonella contamination of eggshells. For this purpose, a total of 4,200 eggs were collected from 28 positive flocks and analyzed according to draft Annex D of International Organization for Standardization Method 6579. Molecular characterization of the Salmonella strains was obtained by the pulsed-field gel electrophoresis method with two restriction enzymes, XbaI and BlnI. The relationship between the presence of Salmonella on eggshells and rearing practices was studied by using multiple correspondence analysis. Results showed that 39.3% of the positive flocks had at least one positive eggshell, with a total of 1.05% of eggshells testing positive for Salmonella. We detected the same serovars on samples taken from the farm and from eggshells within a given flock, with isolates sharing the same genetic pattern in 7 of 11 flocks. Eggshells tested positive for Salmonella in flocks (i) located where delivery trucks pass near air entrances of the poultry house, (ii) with high holding capacity (>30,000 laying hens), and (iii) with more than five positive samples coming from the farm environment, as well as in cases of flocks with a maximum egg-laying rate of >96% and in cases where farmers worked in other animal production. This study provided valuable information that could be used for risk management and risk assessment studies.

Improvements required for the detection of Salmonella Pullorum and Gallinarum.

Detection of the specific Salmonella serovar Gallinarum, which is divided into the biovars Pullorum and Gallinarum, is compulsory under the national hygienic and sanitary control regulations of France for breeding flocks whose offspring are exported. Our aim was to examine the suitability of bacteriologic and serologic methods routinely used in France to screen serum samples and organs for S. Gallinarum. Since bacteriologic reference techniques are designed to isolate the commonly occurring non-typhoid serovars, such as S. Typhimurium, S. Enteritidis, and others that cause outbreaks of foodborne illness, they may not be particularly suitable for detecting S. Pullorum and S. Gallinarum. This hypothesis was confirmed by the inoculation of 10-wk-old chickens and 1-d-old chicks with various strains of S. Pullorum and S. Gallinarum. The most reliable enrichment media were selenite cystine and Rappaport-Vassiliadis broths. Moreover, on the usual plating media, colonies were small, grew more slowly than the common serovars (in 48 h instead of 24 h), and had an unusual appearance. Since the rapid slide agglutination (RSA) test is based only on antigens from standard and variant strains of S. Pullorum, it may not readily detect S. Gallinarum. In our study, it detected infection in all 10-wk-old chickens inoculated with S. Pullorum strains but did not detect any antibodies against S. Gallinarum. Therefore, S. Gallinarum antigens must be added to the S. Pullorum antigens used in the RSA test in order to detect antibodies produced by birds infected with either biovar.

Reliable ELISAs showing differences between resistant and susceptible lines in hens orally inoculated with Salmonella Enteritidis.

Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10(8) CFU S. Enteritidis per animal. Anti-lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line (p = 0.013, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with 1.7 x 10(8) CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM (p = 0.017, 0.017 and p < 0.001 respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.