Effect of feeding dairy calves with milk fermented with selected probiotic strains on occurrence of diarrhoea, carriage of pathogenic and zoonotic microorganisms and growth performance.

Calf-diarrhoea is a major health problem in dairy calves and a primary reason for use of antimicrobials. We aimed to investigate the effect of feeding milk fermented with a combination of four probiotic bacterial strains to young-calves on; occurrence of diarrhoea and associated-pathogens (bacteria, virus and parasites), shedding of Salmonella Dublin and Campylobacter, occurrence of virulence genes linked to Clostridium perfringens, Enterotoxigenic Escherichia coli and shiga-toxin producing E. coli (STEC), as well as growth performance. For this, 143 new-born calves from three Danish dairy-farms were allocated into Treatment- (fed the fermented milk for the first 8-weeks-of-life) and Control-groups (fed regular farm-milk). Diarrhoea was observed in 18.6 % (Farm 1), 22.4 % (Farm 2) and 15.7 % (Farm 3) of the total registrations mainly within the first 3-weeks-of-life. C. perfringens was the most frequently detected pathogen. The treatment did not affect the occurrence of virulence genes linked to STEC and C. perfringens and, overall, their detection levels were very low/undetected. The statistical model applied found no significant effect of the treatment on prevalence of early-diarrhoea (≤ 3 weeks), late-diarrhoea (>3 weeks), occurrence of C. perfringens and Cryptosporidium parvum or levels of Campylobacter spp. Limited detection of the other pathogens and associated virulence-genes under study, did not allow for assessment of the impact of the treatment on their occurrence. Notably, the feeding-approach showed a significant detrimental effect on daily-weight-gain. The inefficacy of the treatment may be associated with the complexity of influencing factors under field conditions including management practices.

Inhibition of Virulence Gene Expression in Salmonella Dublin, Escherichia coli F5 and Clostridium perfringens Associated With Neonatal Calf Diarrhea by Factors Produced by Lactic Acid Bacteria During Fermentation of Cow Milk.

Diarrhea is a major health problem in neonatal and young calves worldwide. It can be caused by a variety of infectious agents, including the bacteria Salmonella enterica serovar Dublin (S. Dublin), enterotoxigenic Escherichia coli (ETEC), and Clostridium perfringens. Preventive alternatives to antibiotic treatment should be identified. As a first step toward this, the aim of the current study was to examine whether cell-free supernatants from cow milk fermented by lactic acid bacteria affects virulence-gene expression in strains of S. Dublin, ETEC E. coli F5 and C. perfringens. pH-neutralized, cell-free, spent medium of milk (nCFSM) fermented by 61 different lactic acid bacteria (LAB) and non-LAB starter cultures belonging to 17 genera was assayed for their effect on expression of important virulence factors (S. Dublin hilA, ssrB, ssaG, flhD, prgI, fliC; ETEC E. coli F5 fanC, estA, fim41a; C. perfringens cpa), when the bacteria were grown in the nCFSM. Screening was done using either a promoter-reporter expression system or RT-qPCR. nCFSM from Bifidobacterium longum BL-15955 and Limosilactobacillus reuteri LR-33016 downregulated the expression of fanC, fim41a and estA genes in the four tested ETEC E. coli F5 strains without affecting their growth, while mainly B. longum BL-15955 downregulated expression of cpa in the four tested strains of C. perfringens. nCFSM from the mixed cultures; NU-TRISH® BY-Mild (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Bifidobacterium BL-15954) and COMBO4 (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), as well as Lactobacillus helveticus CNRZ32 downregulated the tested virulence genes in the three tested strains of S. Dublin. To enable possible downregulation of the expression of virulence genes in all three target bacteria simultaneously, nCFSM was prepared from NU-TRISH® By-Mild in combination with B. longum BL-15955 (i.e. a four-strain combination). The nCFSM from this combination downregulated the virulence genes expression in all the three species. In the future, NU-TRISH® By-Mild and B. longum BL-15955 in combination could potentially be used for prevention of neonatal calf diarrhea caused by S. Dublin, E. coli F5, and C. perfringens, reducing the need for antimicrobial treatment, however, field studies are needed to prove that.

Salmonella Typhimurium Level in Mealworms (Tenebrio molitor) After Exposure to Contaminated Substrate.

Findings of viable Salmonella spp., which are important foodborne pathogens, are seemingly not reported in mealworms (Tenebrio molitor) for feed and food. Still, the bacterial load of mealworms is naturally high and includes members of the Enterobacteriaceae family to which Salmonella belong. This indicates that Salmonella may be able to thrive in mealworms if introduced into the production. Therefore, this study aimed to assess the quantitative level of Salmonella enterica serovar Typhimurium (ST) in mealworms over a 14-day course after exposure to substrate contaminated with ST levels from 1.7 to 7.4 log CFU/g at start (i.e., day 0). The level of ST found in larvae was below the quantitative detection level (1 or 2 log CFU/g) on day 1 in larvae exposed to contamination levels of 1.7, 3.4, and 3.6 log CFU/g opposed to contamination levels of 5.4, 5.6, and 7.4 log CFU/g, respectively. The maximum level of ST detected in individual 1-g larvae samples was 5.8 log CFU/g, but the level varied among the triplicate samples from each sampling, and the highest average value was 5.3 ± 0.3. Beyond day 7, only larvae exposed to the contamination level of 7.4 log CFU/g were >1.0 log CFU/g in the triplicate samples. However, qualitative testing (10 g) showed the presence of ST in larvae until the end of the experiment on day 14 except for the lowest contamination level of 1.7 log CFU/g. Parallel testing of surface disinfected larvae indicated that some larvae may be ST-positive due to Salmonella residing on the surface only. Still, any detection of Salmonella is of concern from a food safety perspective. In substrate with contamination levels below 3.6 log CFU/g, the level of ST was below the quantitative detection limit within a few days. Still, ST was detected until the end of experiment on day 14 except for the lowest contamination level of 1.7 log CFU/g. This study indicates the importance of avoiding introduction of Salmonella into the production, e.g., via contaminated substrate in order to avoid Salmonella-positive larvae as they remained positive for at least 14 days (except at the lowest contamination level).

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

Characterization of Antibiotic Resistance Gene Abundance and Microbiota Composition in Feces of Organic and Conventional Pigs from Four EU Countries.

One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1, sul2, strA, tet(A), tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our expectations, there were no extensive differences between the abundance of tested antibiotic resistance genes in microbiota originating from organic or conventionally housed pigs within individual countries. There were also no differences in the microbiota composition of organic and conventional pigs. The only significant difference was the difference in the abundance of antibiotic resistance genes in the samples from different countries. Fecal microbiota in the samples originating from southern European countries (Italy, France) exhibited significantly higher antibiotic resistance gene abundance than those from northern parts of Europe (Denmark, Sweden). Therefore, the geographical location of the herd influenced the antibiotic resistance in the fecal microbiota more than farm's status as organic or conventional.

Campylobacter contamination and the relative risk of illness from organic broiler meat in comparison with conventional broiler meat.

Danish organic broiler meat, represented by carcasses sampled at the end of processing after chilling, was more frequently contaminated with thermotolerant Campylobacter spp. than conventional broiler carcasses; the yearly mean prevalence being 54.2% (CI: 40.9-67.5) for organic and 19.7% (CI: 14.8-24.7) for conventional carcasses. Campylobacter jejuni was the most frequently isolated species. The difference in prevalence was obvious in all quarters of the year. Contamination of organic and conventional broiler carcasses was more likely to occur in the warmer summer months, in this case in the third quarter, as also documented for conventional broiler flocks. When contaminated, the mean concentration of Campylobacter on neck skin samples of organic and conventional carcasses was not significantly different (P=0.428); 2.0±0.65 log 10 cfu/g and 2.1±0.93 log 10 cfu/g, respectively. Assessing the relative risk of becoming ill following exposure to Campylobacter on conventional or organic broiler meat indicated that the risk per serving from organic carcasses was 1.7 times higher than that of conventional carcasses. The higher risk of illness from organic broiler carcasses compared with conventional broiler carcasses emphasizes the importance of implementing control measures in organic broiler production.

Survival and transmission of Salmonella enterica serovar typhimurium in an outdoor organic pig farming environment.

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g-1) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n=294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n=10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.