Sows affect their piglets' faecal microbiota until fattening but not their Salmonella enterica shedding status.

Recent studies have shown that Salmonella shedding status affects sows' microbiota during gestation and that these modifications are reflected in the faecal microbiota of their piglets at weaning. The aims of this study were: (a) to evaluate the persistence, up to the fattening period, of the previously measured link between the microbiota of piglets and their mothers' Salmonella shedding status; and (b) measure the impact of the measured microbiota variations on their Salmonella excretion at this stage. To achieve this, 76 piglets born from 19 sows for which the faecal microbiota was previously documented, were selected in a multisite production system. The faecal matter of these swine was sampled after 4 weeks, at the fattening stage. The Salmonella shedding status and faecal microbiota of these animals were described using bacteriological and 16S rRNA gene amplicon sequencing respectively. The piglet digestive microbiota association with the Salmonella shedding status of their sows did not persist after weaning and did not affect the risk of Salmonella excretion during fattening, while the birth mother still affected the microbiota of the swine at fattening. This supports the interest in sows as a target for potentially transferrable microbiota modifications.

Salmonella shedding status of the sow affects the microbiota of their piglets at weaning.

AIM: To observe the transfer of the digestive microbiota from sow to piglet, describe the impact of the sow's Salmonella shedding on this transfer and identify transferred populations that could be associated with the future Salmonella status of the piglets. METHODS AND RESULTS: Salmonella shedding status of 19 sows was determined at the beginning and end of gestation. Four piglets were randomly selected from each sow. Using MiSeq, the microbiotas of the sows at the end of gestation and of their piglets 1 day before weaning were described. Results showed that the Salmonella shedding of the sows, the birth mother, the lairage room, the parity and the contamination of the lairage environment were associated to the microbiota of the piglets (permanova P < 0·05). Several genera were associated with piglets born from negative or positive sows. CONCLUSION: There is a link between the microbiota of the sows at the end of gestation and the microbiota of their piglets at weaning, and the Salmonella shedding of the sow is associated with the microbiota of the piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella status of the sows affects the microbiota of their piglets and could affect the long-term Salmonella colonization resistance of these animals and their health.

Feed presentation options in Swine early fattening mitigates Salmonella shedding and specifically modulates the faecal microbiota.

AIMS: The object of this study was to determine the impact of only modifying the processing and/or particle size of pig feed on Salmonella shedding and faecal microbiota. METHODS AND RESULTS: Pigs were fed a diet that varied only by their processing (pellet or mash) and their particle size (500, 750 or 1250 µm) for 21 days. Salmonella detection in faeces and seroconversion were determined. Faecal microbiota was assessed by Ion Torrent amplicon sequencing and real-time PCR. Significantly fewer pigs (P < 0·05) shed Salmonella in the groups fed mash 500 (1) and mash or pellet 1250 (5 each) compared to the commercial reference group (15) fed pellet 500. Both mash processing and large particle size raised the proportion and number of bacteria from the Bifidobacterium genus in the faecal microbiota of the pigs. Thirteen other taxa significantly varied (P < 0·0005) with feed presentation. CONCLUSION: Mash processing and/or large particle size in pig feed reduces Salmonella shedding prevalence and promotes beneficial populations of digestive microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to demonstrate a difference in Salmonella shedding through only modifying pig feed presentation and is the first to extensively describe modifications of faecal microbiota.

Impact of mash feeding versus pellets on propionic/butyric acid levels and on total load in the gastrointestinal tract of growing pigs.

Feed characteristics may influence the bacterial community composition and metabolic activities in the pig gastrointestinal tract, known to be associated with positive effects on the gut. Use of mash feed is associated with reduced excretion, but little is known of its effect on the population or of the mechanism of action. Our objectives were to assess the effect of feed texture combined with feed particle size on VFA profiles and levels, total count, and the presence of genes encoding virulence factors of pathogenic strains in the digestive tract along with their impact on pig performance of fattening pigs. Pigs ( = 840) on a commercial farm received mash or pellet diets of different particle sizes during the fattening period. Caecal and colon contents from 164 pigs were sampled at the slaughterhouse for enumeration of by quantitative PCR (qPCR) and for VFA quantification by capillary gas chromatography. The gene was used to enumerate total . Improved pig performances associated with pellet texture and a 500-µm size were observed. Caecal ( = 0.02) and colon ( < 0.01) propionic acid concentrations were lower for pigs receiving pellet rather than mash feed. Similarly, caecal ( = 0.01) and colon ( < 0.001) butyric acid concentrations were also lower for pigs receiving pellet rather than mash feed, as determined by capillary gas chromatography. Moreover, caecal ( = 0.03) and colon ( < 0.001) butyric acid concentrations were higher for pigs receiving a feed with a 1,250-µm particle size rather than a 500-µm particle size. On the other hand, total caecal and colon levels were higher for pigs receiving pellet feed than for those receiving mash feed. For total enumeration, caecal ( < 0.01) and colon ( < 0.01) gene copies were higher for pigs receiving pellet rather than mash feed. No effect of particle size on fatty acid concentrations or on numbers was observed. Virulence gene quantification revealed no trend. Taken together, results showed that mash feed is associated with lower growth performance but with favorable intestinal changes linked to VFA levels and reduction in the intestine.

Capillary electrophoresis single-strand conformation polymorphism for the monitoring of gastrointestinal microbiota of chicken flocks.

The objective of the present study was to evaluate the capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) to characterize poultry gut microbiota and the ability of this molecular method to detect modifications related to rearing conditions to be used as an epidemiological tool. The V3 region of the 16S rRNA gene was selected as the PCR target. Our results showed that this method provides reproducible data. The microbiota analysis of individuals showed that variability between individual fingerprints was higher for ileum and cloaca than for ceca. However, pooling the samples decreased this variability. To estimate the variability within and between farms, we compared molecular gut patterns of animals from the same hatchery reared under similar conditions and fed the same diet in 2 separate farms. Total aerobic bacteria, coliforms, and lactic acid bacteria were enumerated using conventional bacteriological methods. A significant difference was observed for coliforms present in the ceca and the cloaca depending on the farm. Ileal contents fingerprints were more closely related to those of cloacal contents than to those of ceca contents. When comparing samples from the 2 farms, a specific microbiota was highlighted for each farm. For each gut compartment, the microbiota fingerprints were joined in clusters according to the farm. Thus, this rapid and potentially high-throughput method to obtain gut flora fingerprints is sensitive enough to detect a "farm effect" on the balance of poultry gut microbiota despite the birds being fed the same regimens and reared under similar conditions.

Description and sources of contamination by Campylobacter spp. of river water destined for human consumption in Brittany, France.

Presence or absence of Campylobacter spp. in water of five rivers upstream from an intake point for drinking water production was investigated, and isolates genetically compared with human, pig and poultry isolates in order to determine their source. River water and drinking water obtained from these rivers were sampled one time per month, over a period of one year, and tested for Campylobacter. Isolates were typed by PFGE. Campylobacter was not detected in treated drinking water, but 50% of the river samples were contaminated. Contamination was observed on the four seasons. In total, 297 Campylobacter isolates were collected and generated 46 PFGE profiles. Campylobacter jejuni was the most frequently detected species in samples (74.1% of the isolates), followed by Campylobacter coli (17.8%) and Campylobacter lari (8.1%). Forty-two of the 46 PFGE profiles were unique. Only one genotype was detected three times in a river during the year and four genotypes in two different rivers. When compared to animal and human Campylobacter PFGE profiles, 14, 11 and one Campylobacter genotypes from water were genetically closed to human, poultry, and pig Campylobacter genotypes, respectively. The Campylobacter population displayed a high level of genetic diversity, suggesting that contamination originated from various origins. Human, poultry and pig were sources of contamination of the river by Campylobacter. Finally, no Campylobacter were detected in drinking water, indicating that the risk of outbreaks due to consumption of drinking water is low.

Comparison of genetic profiles of Campylobacter strains isolated from poultry, pig and Campylobacter human infections in Brittany, France.

Five hundred eighty-two Campylobacter isolates (177 from humans, 319 from poultry and 86 from pig) collected in Brittany, France, in 2003 and 2004 were typed by pulsed-field gel electrophoresis. The number of human cases increased during the hot season, particularly for C. jejuni. Twelve genetic groups out of 27 contained human isolates collected over the two years. These groups had 21.3 and 17.0% of the isolates obtained in 2003 and 2004, respectively. In four cases, isolates from 2003 have the same Pulsed-field gel electrophoresis (PFGE) profile as isolates from 2004. Six PFGE profiles common to poultry and human isolates were identified. Poultry isolates were found in 47 clusters containing human isolates. Caeca from farms and slaughterhouses accounted for 66% of these isolates, with chicken legs obtained from supermarkets accounting for the other 34%. Pig isolates never clustered with poultry and human isolates. In conclusion, the analysis of the genetic profiles of Campylobacter resulting from human cases showed that there were few identical or genetically close isolates between the human cases declared in 2003 and those declared in 2004. This highlighted a great genetic diversity in the isolates and indicated that it should be difficult to bind the human infections with groups of Campylobacter isolates presenting particular genetic profiles. The Campylobacter isolates obtained from the two animal production systems had different genotypes, and isolates from pigs differed genetically from isolates obtained from humans. We found that 44.6% of human Campylobacter isolates were genetically related to genotypes found in poultry and a part of these campylobacteriosis are due to contact with poultry. This is not particularly surprising in Brittany, a farming area with many animal-rearing farms and slaughterhouses. This work highlights the implication of the poultry in the French human cases and that handling of poultry is also an important risk for Campylobacter infection in humans.

An integrated approach of genetic resistance to Salmonella carrier state in fowls: from genetics to genomics and modelling.

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

Risk assessment strategies for Europe: integrated safety strategy or final product control: Example of Listeria monocytogenes in processed products from pork meat industry.

The European regulation 2160/2003 of November 17th, 2003 clearly shows the European strategy of zoonosis monitoring and control as an integrated approach, including the entire food production chain with a first application to Salmonella control in different animal species. This regulation is the consequence of a risk assessment performed with a "farm to fork" philosophy. European strategy is scarcely different from the American strategy, despite the fact that both were achieved by a quantitative risk assessment, as for instance, in the USA the control of Salmonella in eggs is supposed to be completed by refrigeration. Nevertheless, the EU will still have a final product control approach towards future regulations on microbiological criteria for foodstuffs. The final production monitoring and control with HACCP (93/43/EC) and microbiological criteria is the only one available for L. monocytogenes in foodstuffs. The purpose of this paper is to discuss alternative control strategies for L. monocytogenes in pig production including integrated risk assessment. In France, most of the food-borne outbreaks associated with L. monocytogenes in delicatessen were due to one particular group of strains belonging to serovar 4b and presenting a particular RFLP/PFGE (Restriction Fragment Length Polymorphism/Pulsed Field Gel Electrophoresis) profile. The outbreak itself is always associated with the initial contamination of a RTE ("ready to eat") product and re-contamination by inappropriate handling after cooking. Consequently, in most cases the RTE product is subject to inadequate refrigeration during an excessive shelf-life. The responsibility of the food industry and the consumer is clearly engaged during this scenario of foodborne diseases. The question is how to avoid the introduction of this particular strain of L. monocytogenes in the food chain. In a study we tried to evaluate the risk of pig carcass contamination at slaughterhouse level and to identify the main risk factors associated with the infection of live pigs. In most cases inappropriate cleaning and disinfection of surfaces were associated with the contamination of raw meat, but in some cases the introduction of epidemic strains in the food chain was also associated with primary production. Feeding with soup in piggeries seemed to select a particular microbial ecology associated to L. monocytogenes contamination of live pigs. The possible strategies that may be used to control L. monocytogenes in live pig production are not yet developed sufficiently to be included in the EC regulation but should be discussed in more detail.

Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to-finish herds.

Fattening-pigs carriers of Salmonella enterica are believed to be a main source of carcass and pork contamination at the later steps of the meat process. We did a prospective study in 2000-2001 in 105 French farrow-to-finish pig farms. In each farm, a batch of contemporary fattening pigs housed in the same room was followed throughout the fattening period. Salmonella shedding was assessed on environmental samples of faecal material (taken by means of pairs of gauze socks) analysed by classical bacteriological methods. 36.2% of the batches studied had at least one contaminated environmental sample and therefore were classified as Salmonella-shedding batches. Logistic regression was used to assess the association between managerial and hygiene practices and health status and the shedding risk at the end of the finishing period. Emptying the pit below the slatted floor after the previous batch of sows was removed and frequent removal of sow dung during the lactation period were protective. Presence of residual Salmonella contamination of the floor and pen partitions in the fattening rooms before loading the growing pigs also was a risk factor. The risk for Salmonella shedding at the end of the fattening period was increased when dry feed (versus wet feed) was provided during the fattening period. Lastly, Lawsonia intracellularis seroconversion and PRRSV seropositivity during the fattening period also was a risk factor for Salmonella shedding.

Listeria spp. contamination in piggeries: comparison of three sites of environmental swabbing for detection and risk factor hypothesis.

Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialized countries. Listeria carriage by pigs at the herd level could be a primary source for carcass contamination. Forty-seven finishing pig facilities were involved in the present study designed to compare three environmental swabbing sites in order to detect Listeria spp. in piggeries. Swabs were taken from the pen walls, the perianal regions of the pigs and the trough/feeder of the piggery premises. Listeria contamination of wet or dry feed given to the pigs was also investigated. The capacity of the various sampling sites for Listeria spp. detection were compared with a maximum likelihood estimation method. Listeria spp. were recovered in 74% of the pens studied and L. monocytogenes was detected in 15% of pens. With a specificity of 99%, sensitivity estimates (and 95% CI) of the Listeria spp. detection method were 93.4% (72.7-98.7) for pen walls, 73.1% (54.9-85.9) for pigs and 66.6% (48.6-80.7) for the trough/feeder. Listeria spp. were isolated from 84% of wet feed samples and 5% of dry feed samples. Listeria monocytogenes was found in 13% of wet feed samples. The type of feeding (wet versus dry) was associated (P < 0.001) with Listeria spp. contamination of both the pen and the feed. The results of this study confirm that Listeria spp., including L. monocytogenes, are present in pig facilities. Pen wall swabbing appears to be an effective way to assess Listeria spp. status of finishing pigs. The type of feeding (wet versus dry) could play a role in pig contamination.

Longitudinal serological responses to Salmonella enterica of growing pigs in a subclinically infected herd.

A longitudinal survey was conducted in France in a subclinically Salmonella-infected farrow-to-finish pig farm to describe the time-course of the serological response to Salmonella enterica in growing pigs. We used three batches of sows and their corresponding litters (n = 31 litters). Among these, 256 pigs randomly selected and individually identified were followed from the first week of age until slaughter. Serial individual blood samples were submitted to indirect Salmonella-ELISA testing. Salmonella shedding was investigated by bacteriological testing of faecal material regularly collected from the sows and pigs and by environment swabs taken from the pens. Caecal contamination was checked at the slaughterhouse. Information about litter composition (filiation), location of the pigs in post-weaning and fattening pens, sanitary events, sex and body weights was recorded. 11.6% of the pigs shed S. enterica; 52% of pigs seroconverted before slaughter. The age-related variation of the natural logarithm of calibrated optical densities (COD) of pigs was described with two linear mixed models. From 8 to 61 days of age, the decrease in COD with age was fitted with a model including random effects of the animal and the dam on the intercept and slope, a batch random effect on the intercept and an individual birth-weight fixed effect on the intercept. The dam random effect was explained by the parity of the sow, Salmonella shedding by the sow during the farrowing phase and the value of the optical density of colostrum collected at parturition. A second model fitting the increase in COD from 61 days of age until slaughter included the random effect on intercept of the batch and the random effects on slope and intercept of the animal, the dam and the pen in which the followed animals were located during the fattening phase and the environmental contamination as fixed effect. In this second model, no relation was found between individual slaughter-bacteriological results and increasing COD values. Considering seroconversion time between 61 days of age and slaughter, survival function were constructed using the Cox proportional-hazards model. Both methods suggested that seroconversions generally occurred during the last third of the fattening phase (from 140 days of age to slaughter), while shedding was observed during the first half of the fattening period. The fitted models suggest the existence of clusters (such as pen and litter of origin).

Feasibility of fluorescent detection of pathogens on pork carcasses.

The direct immunofluorescent detection of pathogens on pork skin is evaluated. Calibrate contamination of pork skin with Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) is developed in 2 h at 4 degrees C. Then a specific indirect immunofluorescent staining protocol is optimized in order to obtain specific and intensive signals able to be detected by electronic cameras (deported microscopy). Despite the individual staining of ST and Lm is possible on pork skin and is specific and bright, the deported microscopy failed to detect these particles. After respectively 3 and 6 h, we obtain micro-colonies of ST and Lm. Due to the limited power of the video camera used, only the microscope permits the detection on the skin. However, our work gives standard conditions to mime the pathogens contamination and staining directly on a biological matrix such as pork skin. This work is a first step in the development of direct and rapid detection of pathogens on biological matrix.

Contamination of pigs by nose-to-nose contact or airborne transmission of Salmonella Typhimurium.

The aim of this study is to assess the risk of contamination by Salmonella Typhimurium of pigs by nose-to-nose contact or the airborne route. Thirty twelve-week-old SPF pigs were divided into 4 groups housed in 4 different rooms: the first room contained Salmonella-free control pigs (n = 4), the second room had 10(3) CFU S. Typhimurium inoculated pigs (n = 5) and non-inoculated "contact" pigs (n = 4), the third room had pigs (n = 8) receiving potentially contaminated air from the following room through a hole (4 pigs housed in the pen situated near the hole and 4 pigs in the pen at the opposite side of the room), and the fourth room had pigs (n = 5) inoculated with 10(6) CFU Salmonella Typhimurium and also non inoculated "contact" pigs (n = 4). The "contact" and the inoculated pigs were housed in adjacent pens allowing nose-to-nose contact. The 5 pigs orally inoculated with 10(6) CFU S. Typhimurium were bacteriologically and serologically positive 1 week later and their environment was contaminated as early as 1 day pi. The faecal samples of 4 nose-to-nose contact pigs were bacteriologically positive and one of them was seropositive 5 weeks pi before the pigs were commingled. The 8 pigs housed in the third room received S. Typhimurium by an active airflow coming from the contaminated room (1000 m3/hour). Their faecal samples remained negative until 8 weeks pi but the environmental swabs taken in the room close to the airinlet were contaminated 2 days pi and positive swabs were found elsewhere in the room 5 weeks pi. Two seropositive pigs were encountered 8 weeks pi in the pen situated near the hole. Only one among the 5 pigs inoculated with 10(3) CFU had bacteriologically positive faeces 1-week pi and the 4 pigs kept in nose-to-nose contact with them remained negative. A dose of 10(3) CFU was too small to induce persistent excretion and to stimulate a humoral immune response. However, the dose of 10(6) CFU induced contamination of nose-to-nose contact pigs and contamination of the environment by airflow.

Evaluation of cellular proliferation on collagenous membranes.

Collagenous membranes are used in guided tissue regeneration designed to repair damage done to the periodontium by disease. The cells primarily responsible for the process are desmodontal fibroblasts. We have studied the behavior and proliferation of fibroblasts grown on collagenous membrane. Fibroblasts grown on plastic served as control. Cellular proliferation was evaluated using a colorimetric test for cytotoxicity and by flow cytometry. Scanning electron microscopy was used to observe cellular morphology. Results showed a reduction in cell number, together with a modification in cell cycle, but good preservation of cellular morphology for cultures grown on collagenous membrane. These results support the in vivo use of collagenous membrane in guided tissue regeneration.

Effect of Porphyromonas gingivalis on epithelial cell MMP-9 type IV collagenase production.

Porphyromonas gingivalis is reportedly capable of stimulating the expression of host cell matrix metalloproteinases (MMP), contributing to tissue destruction. However, the impact of this bacterium on specific molecules remains to be determined. In this study, we evaluate the effect of P. gingivalis on regulation of MMP-9 expression in human gingival epithelial cells (HGEC). Various inocula of P. gingivalis were added to cultures of HGEC. The effects of live bacteria, heat-killed bacteria, and outer membrane extract were analyzed. MMP-9 secretion by HGEC was evaluated by enzyme-linked immunosorbent assay. For inocula smaller than one bacterium per cell, the quantity of MMP-9 secreted by HGEC was increased in comparison to control conditions. For inocula from 2.5 to 250 bacteria per cell, an inhibition of MMP-9 secretion in a dose-response fashion was observed, with a maximum reduction (ranging from 80 to 95% in five experiments) at 50 bacteria per cell. Gelatin zymograms confirmed the decrease in MMP-9 secretion. A band of 83 kDa, corresponding to activated enzyme, was present for inocula of 0.5 to 50 bacteria. Inhibition took place without any alteration of epithelial cell viability. Heat-killed bacteria and outer membrane extract also provoked proenzyme activation but did not inhibit MMP-9 secretion. These results demonstrate a direct effect of P. gingivalis on HGEC, suggesting a specific action on the collagen renewal process at the interface between the epithelium and connective tissue.