Semi-industrial production of a minimally processed infant formula powder using membrane filtration.

Infant formula (IF) is submitted to several heat treatments during production, which can lead to denaturation or aggregation of proteins and promote Maillard reaction. The objective of this study was to investigate innovative minimal processing routes for the production of first-age IF powder, thus ensuring microbial safety with minimal level of protein denaturation. Three nutritionally complete IF powders were produced at a semi-industrial scale based on ingredients obtained by fresh bovine milk microfiltration (0.8 and 0.1-µm pore size membranes). Low-temperature vacuum evaporation (50°C) and spray-drying (inlet and outlet temperatures of 160 and 70°C, respectively) were conducted to produce the T- formula with no additional heat treatment. The T+ formula was produced with a moderate heat treatment (75°C for 2 min) applied before spray-drying, whereas the T+++ formula received successive heat treatments (72°C for 30 s on the milk; 90°C for 2-3 s before evaporation; 85°C for 2 min before spray-drying), thus mimicking commercial powdered IF. Protein denaturation and Maillard reaction products were followed throughout the production steps and the physicochemical properties of the powders were characterized. The 3 IF powders presented satisfactory physical properties in terms of aw, free fat content, glass transition temperature, and solubility index, as well as satisfactory bacteriological quality with a total flora <103 cfu/g and an absence of pathogens when a high level of bacteriological quality of the ingredients was ensured. Protein denaturation occurred mostly during the heat treatments of T+ and T+++ and was limited during the spray-drying process. The IF powder produced without heat treatment (T-) presented a protein denaturation extent (6 ± 4%) significantly lower than that in T+++ (58 ± 0%), but not significantly different from that in T+ (10 ± 4%). Although T- tended to contain less Maillard reaction products than T+ and T+++, the Maillard reaction products did not significantly discriminate the infant formulas in the frame of this work. The present study demonstrated the feasibility of producing at a semi-industrial scale an infant formula being bacteriologically safe and containing a high content of native proteins. Application of a moderate heat treatment before spray-drying could further guarantee the microbiological quality of the IF powders while maintaining a low protein denaturation extent. This study opens up new avenues for the production of minimally processed IF powders.

Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches - mainly antibiotic treatments - are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

Animal board invited review: Improving animal health and welfare in the transition of livestock farming systems: Towards social acceptability and sustainability.

The need to integrate more clearly societal expectations on livestock farming has led the authors of this article to consider that livestock farming systems must be redesigned to position health and welfare at the heart of their objectives. This article proposes a vision of the advances in knowledge required at different scales to contribute to this transformation. After defining health and welfare of animals, the article emphasises the need to consider health in a broader perspective, to deepen the question of positive emotional experiences regarding welfare, and raises the question of how to assess these two elements on farms. The positive interactions between health and welfare are presented. Some possible tensions between them are also discussed, in particular when improving welfare by providing a more stimulating and richer environment such as access to outdoor increases the risk of infectious diseases. Jointly improving health and welfare of animals poses a number of questions at various scales, from the animal level to the production chain. At the animal level, the authors highlight the need to explore: the long-term links between better welfare and physiological balance, the role of microbiota, the psycho-neuro-endocrine mechanisms linking positive mental state and health, and the trade-off between the physiological functions of production, reproduction and immunity. At the farm level, in addition to studying the relationships at the group level between welfare, health and production, the paper supports the idea of co-constructing innovative systems with livestock farmers, as well as analysing the cost, acceptability and impact of improved systems on their working conditions and well-being. At the production chain or territory levels, various questions are raised. These include studying the best strategies to improve animal health and welfare while preserving economic viability, the labelling of products and the consumers' willingness to pay, the consequences of heterogeneity in animal traits on the processing of animal products, and the spatial distribution of livestock farming and the organisation of the production and value chain. At the level of the citizen and consumer, one of the challenges is to better inter-relate sanitary and health perspectives on the one hand, and welfare concerns on the other hand. There is also a need to improve citizens' knowledge on livestock farming, and to develop more intense and constructive exchanges between livestock farmers, the livestock industry and citizens. These difficult issues plead for interdisciplinary and transdisciplinary research involving various scientific disciplines and the different stakeholders, including public policy makers through participatory research.

Heat inactivation partially preserved barrier and immunomodulatory effects of Lactobacillus gasseri LA806 in an in vitro model of bovine mastitis.

Probiotics could help combat infections and reduce antibiotic use. As use of live bacteria is limited in some cases by safety or regulatory concerns, the potential of inactivated bacteria is worth investigating. We evaluated the potential of live and heat-inactivated Lactobacillus gasseri LA806 to counteract Staphylococcus aureus and Escherichia coli infection cycles in an in vitro model of bovine mastitis. We assessed the ability of live and inactivated LA806 to impair pathogen colonisation of bovine mammary epithelial cells (bMECs) and to modulate cytokine expression by pathogen-stimulated bMECs. Live LA806 induced a five-fold decrease in S. aureus adhesion and internalisation (while not affecting E. coli colonisation) and decreased pro-inflammatory cytokine expression by S. aureus-stimulated bMECs (without interfering with the immune response to E. coli). The ability of inactivated LA806 ability to diminish S. aureus colonisation was two-fold lower than that of the live strain, but its anti-inflammatory properties were barely impacted. Even though LA806 effects were impaired after inactivation, both live and inactivated LA806 have barrier and immunomodulatory properties that could be useful to counteract S. aureus colonisation in the bovine mammary gland. As S. aureus is involved in various types of infection, LA806 potential would worth exploring in other contexts.

Molecular characterisation of Staphylococcus aureus strains isolated from small and large ruminants reveals a host rather than tissue specificity.

Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine hosts regardless the locus of isolation (nares and udder). By pulsed-field gel electrophoresis, seven major pulsotypes were identified among 153 isolates recovered from 12 different regions of France as well as from Brazil, the USA and Belgium. Typing of the accessory gene regulator (agr) and capsular (cap) serotype was carried out on all the isolates and revealed the predominance of agr I and III and of cap8 regardless the ruminant host species. Screening for methicilin-resistant S. aureus (MRSA) was carried out by disk diffusion and revealed a prevalence of only 3.2% of MRSA among the strains tested. These results suggest the existence of a host rather than tissue specificity among S. aureus isolates colonising the ruminant species and suggest a limited transmission of those isolates between large (bovine) and small (ovine-caprine) ruminants. The agr class and cap types correlated with pulsotype clusters rather than with a specific host species. Antimicrobial resistance appears not to have contributed to the predominance of any given genotypes, and MRSA prevalence appears very low in ruminant isolates.

Lactobacillus casei BL23 modulates the innate immune response in Staphylococcus aureus-stimulated bovine mammary epithelial cells.

Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1ß and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin ß1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.

A Single Vibrionales 16S rRNA Oligotype Dominates the Intestinal Microbiome in Two Geographically Separated Atlantic cod Populations.

Atlantic cod (Gadus morhua) provides an interesting species for the study of host-microbe interactions because it lacks the MHC II complex that is involved in the presentation of extracellular pathogens. Nonetheless, little is known about the diversity of its microbiome in natural populations. Here, we use high-throughput sequencing of the 16S rRNA V4 region, amplified with the primer design of the Earth Microbiome Project (EMP), to investigate the microbial composition in gut content and mucosa of 22 adult individuals from two coastal populations in Norway, located 470 km apart. We identify a core microbiome of 23 OTUs (97% sequence similarity) in all individuals that comprises 93% of the total number of reads. The most abundant orders are classified as Vibrionales, Fusobacteriales, Clostridiales, and Bacteroidales. While mucosal samples show significantly lower diversity than gut content samples, no differences in OTU community composition are observed between the two geographically separated populations. All specimens share a limited number of abundant OTUs. Moreover, the most abundant OTU consists of a single oligotype (order Vibrionales, genus Photobacterium) that represents nearly 50% of the reads in both locations. Our results suggest that these microbiomes comprise a limited number of species or that the EMP V4 primers do not yield sufficient resolution to confidently separate these communities. Our study contributes to a growing body of literature that shows limited spatial differentiation of the intestinal microbiomes in marine fish based on 16S rRNA sequencing, highlighting the need for multi-gene approaches to provide more insight into the diversity of these communities.

Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus.

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

Control of Colletotrichum acutatum in Strawberry Under Laboratory, Greenhouse, and Field Conditions.

Various fungicides and a heat treatment were assessed for their ability to control strawberry anthracnose caused by the fungus Colletotrichum acutatum under laboratory, greenhouse, and field conditions. The effective dose causing 50% inhibition of mycelial growth (ED50) was 30.5, 12.2, 0.2, 0.15, 0.05, 0.07, and 0.05 µg/ml for the fungicides folpet, captan, propiconazole, difenoconazole, combined prochloraz-Zn/folpet, prochloraz-Zn, and prochloraz-Mn, respectively. In laboratory experiments, infection in segments of strawberry runners treated with pro-chloraz-Zn reached 60%, which was significantly reduced as compared to combined prochloraz-Zn/folpet (90%), captan, folpet, and water controls (100%). In the greenhouse, numbers of naturally infected transplants killed were significantly reduced by all fungicides and the heat treatment (5 min at 49°C) as compared to the non-treated control. Prochloraz-Zn was the most effective chemical control treatment but did not differ significantly from the heat treatment. In field experiments conducted during 1995 and 1996, numbers of naturally infected strawberry transplants killed were significantly reduced by all fungicide treatments relative to the non-treated control. Percent reduction of transplant mortality in the field was 93.3, 93.1, 66.7, 37.7, and 29.1 for prochloraz-Mn, prochloraz-Zn, combined prochloraz-Zn/folpet, propiconazole, and difenoconazole, respectively.

The genome of the generalist plant pathogen Fusarium avenaceum is enriched with genes involved in redox, signaling and secondary metabolism.

Fusarium avenaceum is a fungus commonly isolated from soil and associated with a wide range of host plants. We present here three genome sequences of F. avenaceum, one isolated from barley in Finland and two from spring and winter wheat in Canada. The sizes of the three genomes range from 41.6-43.1 MB, with 13217-13445 predicted protein-coding genes. Whole-genome analysis showed that the three genomes are highly syntenic, and share>95% gene orthologs. Comparative analysis to other sequenced Fusaria shows that F. avenaceum has a very large potential for producing secondary metabolites, with between 75 and 80 key enzymes belonging to the polyketide, non-ribosomal peptide, terpene, alkaloid and indole-diterpene synthase classes. In addition to known metabolites from F. avenaceum, fuscofusarin and JM-47 were detected for the first time in this species. Many protein families are expanded in F. avenaceum, such as transcription factors, and proteins involved in redox reactions and signal transduction, suggesting evolutionary adaptation to a diverse and cosmopolitan ecology. We found that 20% of all predicted proteins were considered to be secreted, supporting a life in the extracellular space during interaction with plant hosts.

Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives.

Staphylococcus aureus is a gram positive opportunistic pathogen and a major concern for both animal and human health worldwide. In some contexts where Lactic Acid Bacteria (LAB) are the normal dominant microbiota, such as in fermented food or in the vaginal ecosystem, S. aureus sometimes colonises, persists, expresses virulence factors and produces food poisoning or urogenital infections, respectively. Studies on the interactions between LAB and S. aureus began a few decades ago and were pursued to shed light on the inhibitory capabilities that LAB might have on S. aureus growth and/or enterotoxin production in fermented foodstuffs. These early studies had the aim of developing methods to prevent staphylococcal food poisoning, thus improving food safety. More recently, the concept of vaginal probiotic LAB has emerged as a promising way to prevent urogenital infections, S. aureus being one of the potential pathogens targeted. This review provides an up-to-date look at the current hypotheses of the mechanisms involved in the inhibition of S. aureus by LAB in both the vaginal ecosystem and in fermented food ecosystems. We also emphasise that post-genomic approaches can now be envisioned in order to study these diverse and complex interactions at the molecular level. Further works in this field will open up new avenues for methods of biocontrol by LAB and/or for biotechnological uses of LAB-compounds to fight against the long-standing, yet incumbent menace of staphylococcal infection.

Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes.

Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.

Molecular physiology of sugar catabolism in Lactococcus lactis IL1403.

The metabolic characteristics of Lactococcus lactis IL1403 were examined on two different growth media with respect to the physiological response to two sugars, glucose and galactose. Analysis of specific metabolic rates indicated that despite significant variations in the rates of both growth and sugar consumption, homolactic fermentation was maintained for all cultures due to the low concentration of either pyruvate-formate lyase or alcohol dehydrogenase. When the ionophore monensin was added to the medium, flux through glycolysis was not increased, suggesting a catabolic flux limitation, which, with the low intracellular concentrations of glycolytic intermediates and high in vivo glycolytic enzyme capacities, may be at the level of sugar transport. To assess transcription, a novel DNA macroarray technology employed RNA labeled in vitro with digoxigenin and detection of hybrids with an alkaline phosphatase-antidigoxigenin conjugate. This method showed that several genes of glycolysis were expressed to higher levels on glucose and that the genes of the mixed-acid pathway were expressed to higher levels on galactose. When rates of enzyme synthesis are compared to transcript concentrations, it can be deduced that some translational regulation occurs with threefold-higher translational efficiency in cells grown on glucose.

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.