Use of probiotic yeasts with biocontrol activity for fermentation of ewe's milk.

BACKGROUND: There are sufficient scienctific studies that support the benefit that fermented dairy products produce in those who consume them. Traditionally, cow's milk has been the most commonly used milk but there is a growing interest in the development of new dairy products, substituting cow's milk with milk from other sources, as well as in the use of microorganisms in fermentation to replace artificial preservatives or treatments that may affect the chemical and organoleptic characteristics of the product. For these reasons, the aim of the present work was to understand the behavior of five potential probiotic yeasts during the fermentation of ewe's milk and to consider their potential use as biocontrol agents. RESULTS: Saccharomyces cerevisiae 3 and Hanseniaspora osmophila 1056 provided the most promising kinetic parameters in the different salt, temperature and pH conditions tested in their technological characterization. The profiles of organic acids and volatile compounds after the fermentation period was noteworthy for contributing to the final aroma of the dairy product. Sensory analysis revealed the sour taste of all samples, and S. cerevisiae 3, Lachancea thermotolerans 1039, and H. osmophila 1056 stood out for an accentuated cheese flavor. In addition, all strains showed biocontrol activity; they reduced the mycelium of the mycotoxigenic molds. CONCLUSION: Saccharomyces cerevisiae 3 and H. osmophila 1056 could be inoculated along with bacterial starters to provide a functional fermented beverage with improved flavor. These strains also have an added value as they act as biocontrol agents. © 2022 Society of Chemical Industry.

Photoperiod Affects Leptin Action on the Choroid Plexus in Ewes Challenged with Lipopolysaccharide-Study on the mRNA Level.

The ovine choroid plexus (ChP) expresses the long isoform of the leptin receptor, which makes this structure a potential target for leptin action. In sheep, leptin concentration in plasma is higher during long days (LD) than short days (SD). This study evaluates the influence a of photoperiod on leptin impact on the gene expression of Toll-like receptor 4 (TLR4), proinflammatory cytokines (IL1B, IL6), their receptors (IL1R1, IL1R2, ILRN, IL6R, IL6ST) and inflammasome components necessary for pro-IL-1ß activation (NLRP3, PYCARD, CASP1), chemokine (CCL2), leptin receptor isoforms (LEPRa, LEPRb) and a suppressor of cytokine signalling (SOCS3) in the ChP of ewes treated or not with lipopolysaccharide (LPS). Studies were conducted on adult female sheep divided into four groups (n = 6 in each): control, leptin (20 µg/kg), LPS (400 ng/kg), and LPS and leptin injected under SD and LD photoperiods. The leptin alone did not affect the gene expression but in co-treatment with LPS increased (p < 0.05) IL1B but only during SD, and SOCS3, IL1R2, IL1RN, IL6ST and CCL2 only during LD, and decreased (p < 0.05) the IL1R1 expression only during SD photoperiod. This indicates that the immunomodulatory action of leptin on the ChP is manifested only under the LPS challenge and is photoperiodically dependent.

The risk of fracture to the tibia from a fragment simulating projectile.

Penetrating injuries due to fragments energised by an explosive event are life threatening and are associated with poor clinical and functional outcomes. The tibia is the long bone most affected in survivors of explosive events, yet the risk of penetrating injury to it has not been quantified. In this study, an injury-risk assessment of penetrating injury to the tibia was conducted using a gas-gun system with a 0.78-g cylindrical fragment simulating projectile. An ovine tibia model was used to generate the injury-risk curves and human cadaveric tests were conducted to validate and scale the results of the ovine model. The impact velocity at 50% risk (±95% confidence intervals) for EF1+, EF2+, EF3+, and EF4+ fractures to the human tibia - using the modified Winquist-Hansen classification - was 271 ± 30, 363 ± 46, 459 ± 102, and 936 ± 182 m/s, respectively. The scaling factor for the impact velocity from cadaveric ovine to human was 2.5. These findings define the protection thresholds to improve the injury outcomes for fragment penetrating injury to the tibia.

Changes in growth hormone secretion and leptin receptor mRNA expression under the influence of leptin and adrenocorticotropin in pituitary cells of early weaned ewe lambs.

Early weaning of ewe lambs strongly stimulates the hypothalamic-pituitary-adrenal axis and is associated with suppressed growth rate despite the increased food intake. At the same time, plasma leptin concentration increases only slightly or undetectably. To better understand this atypical interdependence among somatic stress, leptin, and lamb growth rate, we analyzed impact of leptin and/or adrenocorticotropic hormone (ACTH) on growth hormone (GH) secretion as well as the effect of ACTH on mRNA expression of two splice variants of leptin receptor (LEPRa, LEPRb) in pituitary cells isolated from early weaned ewe lambs. The GH secretion under the influence of leptin and/or ACTH depended on the timing of exposure and hormone concentration. After 6 - 30 h, GH secretion increased under 10-11 - 10-8 M leptin (P ≤ 0.05). However, after 24 - 30 h, GH secretion significantly increased only in cells exposed to both leptin and ACTH compared to culture with leptin only. Simultaneously, there was a significant (P ≤ 0.05) decrease in leptin receptor mRNA expression under the influence of ACTH at 10-8 - 10-6 M after 12 - 30 and 24 - 30 h for LEPRa and LEPRb, respectively. ACTH-related downregulation of LEPR mRNA was associated with a significant (P ≤ 0.05) reduction in leptin-stimulated GH secretion, also after 24 - 30 hours. Thus, the timing of ACTH exposure, followed by decreased leptin receptor mRNA, converged with the timing of decreased GH secretion under the influence of leptin with ACTH. The ACTH-induced downregulation of LEPR mRNA therefore may underlie the decrease in GH. These results show a direct role for leptin, ACTH, and leptin receptor expression in modulation of pituitary GH secretion in early weaned ewe lambs. During the early weaning-induced stress response, the ACTH-mediated decrease in sensitivity of pituitary cells to leptin may abolish a stimulatory effect of leptin on GH secretion and explain in part, the reduction in lamb growth rate.

Multiple microbial cell-free extracts improve the microbiological, biochemical and sensory features of ewes' milk cheese.

This study used cell-free enzyme (CFE) extracts from Lactobacillus casei, Hafnia alvei, Debaryomyces hansenii and Saccharomyces cerevisiae to condition or accelerate Pecorino-type cheese ripening. Compositional, microbiological, and biochemical analyses were performed, and volatile and sensory profiles were obtained. Lactobacilli and cocci increased during ripening, especially in cheeses containing CFE from L. casei, H. alvei and D. hansenii (LHD-C) and L. casei, H. alvei and S. cerevisiae (LHS-C). Compared to control cheese (CC), several enzymatic activities were higher (P < 0.05) in CFE-supplemented cheeses. Compared to the CC (1907 mg kg-1 of cheese), the free amino acid level increased (P < 0.05) in CFE-supplemented cheeses, ranging from approximately 2575 (LHS-C) to 5720 (LHD-C) mg kg-1 of cheese after 60 days of CFE-supplemented ripening. As shown by GC/MS analysis, the levels of several volatile organic compounds were significantly (P < 0.05) lower in CC than in CFE-supplemented cheeses. All cheeses manufactured by adding multiple CFEs exhibited higher scores (P < 0.05) for internal structure, acid taste and juiciness than CC samples. This study shows the possibility of producing ewes' milk cheese with standardized characteristics and improved flavor intensity in a relatively short time.

Antarctic rocks from continental Antarctica as source of potential human opportunistic fungi.

We assessed the diversity of culturable fungi associated with rocks of continental Antarctica to evaluate their physiological opportunistic virulence potential in vitro. The seventy fungal isolates obtained were identified as nine species of Acremonium, Byssochlamys, Cladosporium, Debaryomyces, Penicillium, and Rhodotorula. Acremonium sp., D. hansenii, P. chrysogenum, P. citrinum, P. tardochrysogenum, and R. mucilaginosa were able to grow at 37 °C; in addition, B. spectabilis displayed a high level of growth at 37 and 45 °C. Thirty-one isolates of P. chrysogenum, P. citrinum, and P. tardochrysogenum were able to produce partial haemolysis on blood agar at 37 °C. Acremonium sp., P. citrinum, and P. tardochrysogenum showed spore sizes ranging from 2.81 to 5.13 µm diameters at 37 °C. Of these, P. chrysogenum and P. tardochrysogenum displayed macro- and micro morphological polymorphism. Our results suggest that rocks of the ultra-extreme cold and dry environment of Antarctica harbour cryptic fungi phylogenetically close to opportunistic pathogenic and mycotoxigenic taxa with physiologic virulence characteristics in vitro.

Pathways and Genes Associated with Immune Dysfunction in Sheep Paratuberculosis.

Multibacillary and paucibacillary paratuberculosis are both caused by Mycobacterium avium subspecies paratuberculosis. Multibacillary lesions are composed largely of infected epithelioid macrophages and paucibacillary lesions contain T cells but few bacteria. Multibacillary disease is similar to human lepromatous leprosy, with variable/high levels of antibody and a dysfunctional immune response. Animals with paucibacillary disease have high cell-mediated immunity and variable levels of antibody. This study aims to characterize the immunological dysfunction using TruSeq analysis of the ileocaecal lymph node that drains disease lesions. Immune dysfunction is highlighted by repression of TCR/CD3 genes, T cell co-receptors/co-stimulators, T cell activation and signal-transduction genes. Inflammation was an acute phase response and chronic inflammation, with little evidence of acute inflammation. The high levels of immunoglobulin and plasma cell transcripts is consistent with the anti-MAP antibody responses in paratuberculosis sheep. Also notable was the overwhelming reduction in mast cell transcripts, potentially affecting DC activation of the immune response. This study also shows that there were no fundamental differences in the gene expression patterns in multibacillary and paucibacillary disease, no shift in T cell genes from Th1 to Th2 pattern but rather an incremental decline into immune dysfunction leading to multibacillary pathology.

Performance, metabolic variables and enteric methane production of Santa Inês hair lambs fed Orbignya phalerata and Combretum leprosum.

In this study, the possibility of using Babassu (Orbignya phalerata) and Mofumbo (Combretum leprosum) leaves for lambs feed was verified. Performance, biochemical and haematological parameters, microbial protein synthesis, nutrient apparent digestibility and enteric production of methane (CH4 ) were evaluated. The experimental treatments included diets with forage-to-concentrate ratios of 50:50, with the leaves of the experimental plants replacing 33% of the Cynodon dactylon (Tifton-85) hay with three treatments: control (no hay replacement) and substitution with Babassu or Mofumbo. For the performance study, 24 Santa Inês lambs were used, in a randomized experimental design with eight repetitions (5 male and 3 female) for each treatment and 48 days of experimental trial. After this period, for nine days, six animals from each treatment were allocated in metabolic cages to determine the apparent nutrient digestibility, microbial protein synthesis and nitrogen balance. Simultaneously, enteric CH4 was measured in vivo. The control group showed higher (p < 0.05) apparent digestibility of acid detergent fibre. Enteric CH4 production of lambs fed Mofumbo leaves did not differ from that of the control group, but was lower (p < 0.05) than in those fed with Babassu. The inclusion of Babassu and Mofumbo leaves showed no negative effects on animal health and did not compromise performance. Mofumbo also presented CH4 mitigating potential, indicating that those plants can be used as ingredients in the composition of lamb diets with the advantage of reducing methane production.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn's disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Arginine, the common substrate for production of nitric oxide (NO) and polyamines in mammals, increases in the uterine lumen during the peri-implantation period of pregnancy. However, functional roles of arginine within the uterine lumen for conceptus (embryo and extraembryonic membranes) development have not been elucidated in vivo. To assess roles of arginine in reproductive tissue for survival and development of the conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown of SLC7A1 mRNA, the arginine transporter in ovine conceptus trophectoderm (Tr). Translational knockdown of SLC7A1 mRNA resulted in retarded conceptus development and abnormal function compared to MAO control. Use of MAO-SLC7A1 knockdown in conceptuses decreased arginine transport (73%, P<0.01), the abundance of ornithine decarboxylase, and nitric oxide synthase (NOS3) proteins, arginine-related amino acids [citrulline (76%, P<0.05) and ornithine (40%, P<0.05)], and polyamines, which likely accounts for their retarded development. Also, no alternative arginine precursors (glutamine and glutamate), isoforms of nitric oxide synthase (NOS1 and NOS2), or alternative pathways for polyamine biosynthesis via arginine decarboxylase and agmatinase were activated to rescue conceptus development. Collectively, SLC7A1 is the key transporter of arginine by conceptus Tr, and arginine is essential for conceptus survival and development.-Wang, X., Frank, J. W., Little, D. R., Dunlap, K. A., Satterfield, M. C., Burghardt, R. C., Hansen, T. R., Wu, G., and Bazer, F. W. Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Yeast species and genetic heterogeneity within Debaryomyces hansenii along the ripening process of traditional ewes' and goats' cheeses.

The yeasts present during the ripening process of ewes' and goats' cheeses produced in a small traditional dairy in Mediterranean Spain were isolated and identified. Five hundred and thirty strains pertaining to eleven yeast species representing eight genera were identified using molecular methods. Debaryomyces hansenii was the yeast species most frequently isolated in all cheeses. Other yeast species commonly found in dairy products were present at the first maturing weeks. Two yeast species Trichosporon coremiiforme and Trichosporon domesticum have been reported in cheeses for the first time, and Meyerozyma guilliermondii has been newly isolated from goats' cheeses. Yeast species composition changed greatly along the process; although, D. hansenii dominated at the end of ripening in all cheeses. Most yeast isolates were able to hydrolyze casein and fatty acid esters. One hundred and eighty seven D. hansenii isolates were genotyped by PCR amplification of M13 satellites and an UPGMA dendrogram was constructed. The majority of isolates were grouped in 5 clusters while 7 profiles were represented by 1-3 isolates. These results demonstrate the genetic heterogeneity present in D. hansenii strains isolated from raw milk cheeses.

In vitro thalidomide does not interfere with the activation of complement by M. leprae.

Erythema nodosum leprosum (ENL) is an inflammatory reaction that may occur in multibacillary leprosy patients, and thalidomide is the treatment of choice. Its cause and the mechanism by which thalidomide suppresses ENL are not known. In the skin lesions, im- mune complexes and split products of complement are found. The activation of complement could precipitate ENL, and thalidomide could suppress the inflammation by inhibiting the activation of complement. To determine if thalidomide could suppress the activation of complement, we first incubated normal serum with thalidomide and with M. leprae or zymosan. The amount of residual functional complement was then assessed by determining the dilution of serum required to lyses sheep erythrocytes sensitized by rabbit antibodies (CH50 Assay). M. leprae and zymosan activated complement. The residual complement activity in the serum incubated with M. leprae or with zymosan was equivalent to that incubated with M. leprae or zymosan in the presence of thalidomide, hydrolyzed thalidomide and metabolites of thalidomide. Thalidomide did not inhibit the activation of complement by zymosan, a known initiator of complement activation by the alternative pathway, or by M. leprae.

The plague of the Philistines and other pestilences in the Ancient World: exploring relations between the religious-literary tradition, artistic evidence and scientific proof.

In ancient times the term pestilence referred not only to infectious disease caused by Yersinia pestis, but also to several different epidemics. We explore the relations between references in the Bible and recent scientific evidence concerning some infectious diseases, especially the so-called Plague of the Philistines and leprosy. In addition, some considerations regarding possible connections among likely infectious epidemic diseases and the Ten Plagues of Egypt are reported. Evidence suggesting the presence of the rat in the Nile Valley in the II millennium BC is shown; a possible role of the rat in the plague spreading already in this historical period should be confirmed by these data. While the biblical tale in the Book of Samuel may well report an epidemic event resembling the plague, as to date this infectious disease remains unknown, it is not conceivable to confirm the presence of leprosy in the same age, because the little palaeopathologic evidence of the latter disease, in the geographic area corresponding to Egypt and Palestine, is late, dating back only to the II century AD.

Effect of Bacillus licheniformis and Bacillus subtilis supplementation of ewe's feed on sheep milk production and young lamb mortality.

The purpose of this pilot study was to evaluate under field conditions the effect of a probiotic containing Bacillus licheniformis and Bacillus subtilis on young lamb mortality and sheep milk production when administered in the late pregnancy and lactation feed of ewes. In a sheep farm, two groups of milking ewes with identical genetic material, management, nutrition, health status and similar production characteristics were formed. One group (46 ewes) served as control, while the other one (48 ewes) served as a probiotic-treated group. Both groups of ewes received a similar feeding regiment, but the ewes of the second group were additionally offered a probiotic product containing B. licheniformis and B. subtilis (BioPlus 2B, Chr. Hansen, Denmark) at the approximate dose of 2.56 x 10(9) viable spores per ewe per day. Lamb mortality during the 1.5 months suckling period, and milk yield during the 2 months of milk collection for commercial purposes have been recorded. In the non-treated control group, 13.1% mortality was observed versus 7.8% in the probiotic-treated group (P = 0.33), with mortality being mainly due to diarrhoea. Microbiological examination of diarrhoeic faeces from some of the dead lambs in both groups revealed the presence of Escherichia coli. The average daily milk yield per ewe was significantly lower in the control group (0.80 l) than that in the probiotic-treated group (0.93 l) (P < 0.05). Fat and protein content of milk in ewes that received probiotics was significantly (P < 0.05) increased compared with untreated ewes. It was concluded that supplementing ewe's feed with probiotics may have beneficial effect on subsequent milk yields, fat and protein content.

Identification and characterization of an immunogenic 22 kDa exported protein of Mycobacterium avium subspecies paratuberculosis.

An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 316F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 316F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.

Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi.

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.

Lymphocytic neuritis of the ileum in sheep with naturally acquired and experimental paratuberculosis.

In a comparison of natural and experimentally induced ovine paratuberculosis, aggregations of mononuclear inflammatory cells were detected around nerves in the ileal submucosa in eight of 12 sheep with natural disease and in five of 14 animals with unequivocal experimental paratuberculosis. Such lesions were not seen in 10 other sheep that, despite attempted experimental infection, remained free from the disease, as judged clinically, histopathologically and by PCR assay. The lesions described resembled those observed in human leprosy.