An Overview of the Use and Applications of Limosilactobacillus fermentum in Broiler Chickens.

The implementation of government regulations on antibiotic use, along with the public's concern for drug resistance, has strengthened interest in developing alternatives not only aimed at preserving animal production but also at reducing the effects of pathogenic infections. Probiotics, in particular, are considered microorganisms that induce health benefits in the host after consumption of adequate amounts; they have been established as a potential strategy for improving growth, especially by stimulating intestinal homeostasis. Probiotics are commonly associated with lactic acid bacteria, and Limosilactobacillus fermentum is a well-studied species recognized for its favorable characteristics, including adhesion to epithelial cells, production of antimicrobial compounds, and activation of receptors that prompt the transcription of immune-associated genes. Recently, this species has been used in animal production. Different studies have shown that the application of L. fermentum strains not only improves the intestinal ecosystem but also reduces the effects caused by potentially pathogenic microorganisms. These studies have also revealed key insights into the mechanisms behind the actions exerted by this probiotic. In this manuscript, we aim to provide a concise overview of the effects of L. fermentum administration on broiler chicken health and performance.

Fowl adenovirus-induced different manifestations of the disease in two consecutive chicken breeding flocks in a poultry hall.

This study investigated an adenovirus infection in two consecutive breeding flocks in the same poultry hall. Thirty-six thousand one-day-old chickens of the ROSS 308 hybrid broiler type were kept together in one hall. The chickens in the first breeding flock during fattening did not show any clinical signs of the disease or increased mortality. Typical clinical signs of the adenovirus infection were seen in the second breeding flock. The signs included: depression, apathy, somnolence, a crouched position with a droopy head, fuzzy feathers, anaemic combs and wattles, sporadic nervous signs, and reduced weight gain. Increased mortality was recorded from 18 to 25 days of age, the higher mortality rate resulted from dehydration and exhaustion. The surviving chickens showed growth slightly below average by the end of the fattening period. The necropsies of the chickens in the first flock showed characteristic lesions for inclusion body hepatitis (IBH). Adenoviral gizzard erosions (AGE) were found mainly in the chickens of the second consecutive breeding flock. In both breeding flocks, FAdV-A was detected by polymerase chain reaction (PCR) in the liver and gizzard samples. The presence of fowl adenovirus B was not confirmed in the evaluated samples. The results showed lesions in the first flock typical for IBH, whereas the pathological changes in the second flock were characteristic of AGE.

The Effect of Enterococcus faecium AL41 on the Acute Phase Proteins and Selected Mucosal Immune Molecules in Broiler Chickens.

Probiotic bacteria, including the Enterococcus faecium strain, can improve intestinal mucosal health by several mechanisms, including modulation of the immune response, as well as by improving the protective function of the epithelial barrier. In this study, we tested the effect of Enterococcus faecium AL41 on the acute phase proteins response (blood), gene expression of selected molecules of mucosal immunity (immunoglobulin A, mucin-2, insulin-like growth factor 2) and mucus production (all parts of the small intestine) in broilers. Eighty broiler chicks were divided into two groups: a control and E. faecium AL41 (birds were inoculated with AL41 for 7 days) group. The whole experiment lasted 11 days. Our results revealed that the administration of E. faecium AL41 had no substantial effect on the concentrations of acute phase proteins, but we recorded a significant increase in ß- and γ-globulin fractions at the end of the experiment, which may indicate an improvement in the immune status. A significant prolonged stimulatory effect of E. faecium AL41 on the relative expression of molecules (immunoglobulin A, mucin-2) as well as on the dynamic of mucus production in the chicken intestine was observed. In addition, AL41 significantly reduced the total number of enterococci in the cecum and faeces.

Influence of Immune Parameters after Enterococcus faecium AL41 Administration and Salmonella Infection in Chickens.

Immune response of day-old chicks infected with Salmonella Enteritidis PT4 and preventive administration of Enterococcus faecium AL41 were studied using hematology and flow cytometry of immunocompetent cells in blood, cecum, bursa and spleen for 11 days, and included 220 animals divided into four groups (n = 55). E. faecium AL41 was administered for 7 days to EF and EFSE groups and on day 4 SE and EFSE groups were infected with Salmonella Enteritidis. Values of monocytes at 4 dpi significantly increased in EFSE and lymphocytes at 7 dpi in EF groups. Blood CD3, CD4, CD8 and IgM lymphocytes improved in EF and EFSE groups and IgA in EF group at 4 dpi. Phagocytic activity of probiotic groups was improved in both samples. Cecal IEL and LPL lymphocytes showed at 7 dpi stimulation of CD3, CD4 and CD8 subpopulations in probiotic groups, especially in EFSE group, IgA IEL and IgA with IgM LPL in EF groups. Bursa Fabricii at 7 dpi presented overstimulation of IgG subpopulation in SE group, spleen CD3 and CD8 in EF and EFSE groups. E. faecium AL41 revealed the protective effect and positive influence on the local and systemic immune response in Salmonella Enteritidis PT4 infected chickens.

Production of Intestinal Mucins, sIgA, and Metallothionein after Administration of Zinc and Infection of Ascaridia galli in Chickens: Preliminary Data.

The effect of inorganic zinc and Ascaridia galli infection was studied on MUC1, MUC2 (mucin), sIgA (secretory immunoglobulin A), and metallothionein in the intestines of broilers. Thirty-five-day-old chickens (n = 24), COBB 500 breed, were included in a 14-day experiment. Chickens were divided into 4 groups of 6 chickens each: control ©, Ascaridia galli (AG), Zinc group (Zn), and combined group (AG + Zn). Samples from the intestine for determination of MUC1, MUC2, sIgA, and metallothionein were taken at 7 and 14 days during necropsy. Samples from the jejunum for determination of MUC1, MUC2, sIgA, and metallothionein were taken at 7 and 14 days during necropsy. The results demonstrated that 12 days' administration of inorganic zinc increased production of MUC1 (p < 0.0001) and MUC2 (p < 0.001) in the Ascaridia galli-infected group (Ag + Zn) in comparison to control (C). The beneficial effect of zinc was also revealed in the production of sIgA (p < 0.0001) in the combined group (AG + Zn) at 7 days. The concentration of metallothionein increased mainly in the zinc group (p < 0.01) of first sampling and was upregulated in Zn and AG + Zn groups. The obtained data indicate the use of inorganic zinc as a suitable immunomodulator of intestinal immunity in Ascaridia galli-infected chickens.

The Probiotic Lactobacillus fermentum Biocenol CCM 7514 Moderates Campylobacter jejuni-Induced Body Weight Impairment by Improving Gut Morphometry and Regulating Cecal Cytokine Abundance in Broiler Chickens.

This research was conducted to investigate if the administration of the probiotic Lactobacillus fermentum could influence body weight, intestinal morphometry and the cecal cytokine response in Campylobacter jejuni-infected chickens. Seventy-two 1-day old COBB 500 male chicks were allocated randomly into four experimental groups. (I) Control group (C), in which chicks were left untreated. (II) LB group, treated with L. fermentum. (III) Cj group, infected with C. jejuni and (IV) coexposure group in which both bacteria were administered. Body weight was registered and then all birds were slaughtered; samples from the small intestine and caecum were collected at 4- and 7-days post infection. The experiment lasted eleven days. Villi height and crypt depth ratios of the duodenum, jejunum and ileum were evaluated using appropriate software, while reverse transcription quantitative PCR (RT-qPCR) was utilized for assessing transcript levels of key cecal inflammatory cytokines (IL-1ß, IL-18, IL-17, IL-15, IL13 and IL-4). Campylobacter-infected birds showed lower body weight values than those supplemented with the probiotic; these birds, in turn, proved to be heavier than those reared under control conditions. L. fermentum administration improved morphometrical parameters of the duodenum, jejunum and ileum; in general, villi were larger and crypts deeper than those identified in control conditions. Moreover, the negative effects elicited by C. jejuni were not observed in chickens exposed to the probiotic. Significant differences were also determined with regards to transcript abundance of all evaluated cytokines in the caecum. C. jejuni induced a downregulation of the studied interleukins; however, such a response was heightened by administration of L. fermentum, with an increase rate of transcription that promoted a more effective response to a C. jejuni infection. The effects of experimental treatments proved to vary between sampling points. Conclusively, these results demonstrate that L. fermentum lessens the negative effects elicited by C. jejuni on body weight by alleviating the impact on intestinal morphometry and cecal cytokine response, which ultimately improve chicken growth performance.

Effects of Lactobacillus Fermentum Supplementation on Body Weight and Pro-Inflammatory Cytokine Expression in Campylobacter Jejuni-Challenged Chickens.

Due to the interest in using probiotic bacteria in poultry production, this research was focused on evaluating the effects of Lactobacillus fermentum Biocenol CCM 7514 administration on body weight gain and cytokine gene expression in chickens challenged with Campylobacter jejuni. One-hundred and eight 1-day old COBB 500 broiler chickens were equally assigned to four experimental groups at random. In the control group (C) chicks were left untreated, whereas in groups LB and LBCj a suspension of L. fermentum was administered. A suspension of C. jejuni was subsequently applied to groups Cj and LBCj. Body weight was registered, and the individuals were later slaughtered; cecum samples were collected at 12, 36 and 48 h post-infection (hpi). The entire experiment lasted seven days. Reverse transcription quantitative PCR (RT-qPCR) was used to determine expression levels of IL-1ß, IL-15, IL-17, and IL-18 at each time point. Pathogen-infected individuals were observed to weigh significantly less than those fed with the probiotic. Significant differences were also found in transcript abundance; expression of IL-15 was downregulated by the probiotic and upregulated by C. jejuni. The effects of bacterial treatments were time-dependent, as the expression profiles differed at later stages. The present outcomes demonstrate that L. fermentum both reduces the impact of C. jejuni infection on chicken body weight and regulates positively pro-inflammatory cytokine expression, which ultimately increase bird well-being and improves production.

Lactobacillus fermentum Administration Modulates Cytokine Expression and Lymphocyte Subpopulation Levels in Broiler Chickens Challenged with Campylobacter coli.

This investigation was performed to assess the supplementation of probiotics on cytokine expression and lymphocyte subpopulation in Campylobacter coli challenged chickens. Thirty-six individuals were equally separated into four experimental treatments: C = untreated chickens, LB = probiotic control (Lactobacillus fermentum), Cc = Campylobacter-challenged control, LBCc = probiotic + Cc. All chicks were slaughtered and cecum samples were collected on day 4 postinfection. Gene expression analysis, using reverse transcription quantitative PCR (RT-qPCR), revealed significant differences in cytokine transcript expression between untreated and probiotic-treated chickens. In addition, flow cytometry was used to quantitate the levels of lymphocyte subpopulations. Principal component analysis showed that probiotic administration induced an overall downregulation of cytokine expression. C. coli exposure provoked a similar response to that of L. fermentum but to a lesser extent. Colonization of C. coli in the presence of the probiotic evoked a complex response with an upregulation of some type II cytokines, including interleukin IL-4 and IL-13, which could explain the increased presence of antibodies in both lamina propria and epithelium. Moreover, despite that the percentage of CD8 intraepithelial lymphocytes (IELs) was found to be higher, downregulation of proinflammatory cytokines IL-15, IL-16, and interferon γ was observed. This suggests that the detected CD8 are not effector cells but induced IELs, which release antimicrobial peptides, and are ready to be primed upon encountering antigen. These outcomes demonstrate that probiotic administration promotes a humoral response to a C. coli infection while dampening any potential inflammation mediated by effector T cells in 1-week-old chicks.

Cytokine response after stimulation of culture cells by zinc and probiotic strain.

Intestinal porcine epithelial cells were used for an in vitro analysis of mRNA expression levels of inflammatory cytokines (IL-8, IL-18) and transcriptional factors (MyD88 and NF-κß). Cells were exposed to inorganic and organic zinc sources (in two different concentrations-50 µmol/L and 100 µmol/L) alone or combined with Lactobacillus reuteri B6/1, which was also applied individually. The total exposure time was 4 h. Quantitative reverse transcriptase PCR was used to determine expression levels of the aforementioned parameters. In general, upregulation was observed; however, a decrease of some mRNA's abundance was also determined. Differences in expression were analysed statistically using ANOVA and Tukey analyses. High relative expression was shown for IL-8, IL-18 and MyD88 in groups treated with 100 µmol/L of inorganic sources of zinc (ZnSO4) (p < 0.05), while groups treated with the organic form did not exhibit significant changes in expression. Also, 50 µmol/L of either zinc source did not significantly modify the transcriptional profile of the cytokines and transcription factors, showing that even inorganic sources, at lower concentrations, do not elicit a significant inflammatory reaction. In summary, supplementation of organic zinc source (Gly-Zn chelate) ensures that IL-8, IL-18, MyD88 and NF-κß expression levels are not positively regulated. In contrast, inorganic sources of zinc (ZnSO4) could induce an inflammatory reaction. However, this response could be dampened if L. reuteri B6/1 is administered, showing the helpful aspect of using probiotics to modulate an inflammatory response. Conclusively, the use Gly-Zn chelate appears as an optimal alternative for Zn administration that does not compromise normal intestinal homeostasis.

Influence of oral application of Enterococcus faecium AL41 on TGF-ß4 and IL-17 expression and immunocompetent cell distribution in chickens challenged with Campylobacter jejuni.

Campylobacteriosis is mainly caused by infection with Campylobacter jejuni following consumption or handling of Campylobacter-contaminated poultry meat. The aim of this study was to investigate the effect of probiotic Enterococcus faecium AL41 on TGF-ß4 and IL-17 expression and on immunocompetent cell distribution after C. jejuni infection in broiler chicken, as a second part of the previous study of Karaffová et al. (2017). Accordingly, day-old chicks were randomly divided into four experimental groups of 10 chicks each (n = 10): control (C), E. faecium AL41 (EFAL41), C. jejuni CCM6191 (CJ), and combined E. faecium AL41 + C. jejuni CCM6191 (EFAL41 + CJ). Samples from the caecum were collected on days 4 and 7 post Campylobacter infection (dpi), for the isolation of mRNA of TGF-ß4, IL-17 and for immunohistochemistry. The relative mRNA expression of TGF-ß4 was upregulated in the combined (EFAL41 + CJ) group compared to other groups during both samplings, but the expression of IL-17 was downregulated. Similarly, the highest density of CD3+ was detected in the combined group at 7 dpi, but the number of IgA+ cells was increased in both groups with EFAL41. It was concluded that the EFAL41 probiotic E. faecium strain can modulate the expression of selected cytokines (upregulation of TGF-ß4 but downregulation of IL-17 relative expression), and activate IgA-producing cells in the caeca of chicks infected with C. jejuni CCM6191.

Interaction of TGF-ß4 and IL-17 with IgA secretion in the intestine of chickens fed with E. faecium AL41 and challenged with S. Enteritidis.

The relative mRNA expression of IgA, TGF-ß4, IL-17, and concentration of secretory IgA (sIgA) in small intestine of chickens pretreated with Enterococcus faecium AL41 and challenged with Salmonella Enteritidis PT4 were studied. Salmonella-free day-old chicks (40) Cobb 500 breed, were divided into four groups of 10 chicks each (n = 10): control (C), treated with E. faecium AL41 strain (EFAL41), challenged with Salmonella Enteritidis PT4 (SE), and combined (EFAL41+SE). Expression of IgA and sIgA concentration was upregulated in EFAL41 group in jejunum and ileum on 4 days post-Salmonella infection (dpi). Chicks in combined group demonstrated upregulation of cytokines and IgA expression, and increased sIgA concentration in the intestine flush on 7 dpi. The experiment demonstrated beneficial effect of E. faecium AL41 on IgA production and secretion in intestine. Findings also indicated that IgA played important role in decrease of S. Enteritidis in the intestine, and cytokines TGF-ß4 and IL-17 contributed to the increased IgA secretion.

Dynamics of antibody response and viraemia following natural infection of porcine circovirus 2 (PCV-2) in a conventional pig herd.

Porcine circovirus 2 (PCV-2) is a primary agent of post-weaning multi-systemic wasting syndrome (PMWS), ubiquitous in pig herds. The course of viraemia and seroconversion in naturally infected pigs were investigated in piglets from the 2nd week of their life. Piglets were divided into seropositive (Ab(+)) and seronegative (Ab(-)) groups. Subsequently, after vaccination against PCV-2 (Ingelvac(®) CIRCOFLEX™, Böehringer Ingelheim), they were further divided into non-vaccinated seronegative (NVAC/Ab(-)) and seropositive (NVAC/Ab(+)), and vaccinated seronegative (VAC/Ab(-)) and seropositive (VAC/Ab(+)). PCV-2 colostral antibodies failed to prevent development of natural PCV-2 infection in conventional piglets; however, this occurred at a higher age in comparison with seronegative pigs. Neither colostral nor post-infection antibodies prevented development of viraemia, which persisted up to the end of the study (the 19th week), but without clinical signs of PMWS. Vaccination failed to prevent development of natural PCV-2 infection, but viraemia was limited to between the 8th and 10th week. The presence of colostral anti-PCV-2 antibodies did not show any untoward effect to vaccination; on the contrary, VAC/Ab(+) animals showed the lowest titre of viraemia.

Partial primary deficiency of insulin-like growth factor (IGF)-I activity associated with IGF1 mutation demonstrates its critical role in growth and brain development.

CONTEXT: IGF-I is essential for fetal and postnatal development. Only three IGF1 defects leading to dramatic loss of binding to its type 1 receptor, IGF-1R, have been reported. PATIENT: We describe a very lean boy who has intrauterine growth restriction and progressive postnatal growth failure associated with normal hearing, microcephaly, and mild intellectual impairment. He had markedly reduced concentrations of IGF-I, with IGFBP-3 and ALS serum levels in the upper normal range or above. IGF-I serum concentrations differed according to the immunoassay used. A higher than average GH dose was required for catch-up growth. Given the mismatch between IGF-I and IGFBP-3 levels, we sequenced his IGF1 gene. RESULT: We identified a homozygous missense IGF1 mutation. This causes the replacement of a highly conserved amino acid (arginine 36) by a glutamine (R36Q) in the C domain of the predicted peptide. We showed that the abnormal IGF-I peptide has reduced mitogenic activity and partial loss of binding to its receptor IGF-1R. The patient's IGF-I level was undetectable in a highly specific monoclonal assay but elevated in a polyclonal assay. CONCLUSION: This first report of mild deficiency of IGF-I activity demonstrates that the integrity of IGF-I signaling is important for normal growth and brain development. Molecular defects leading to partial loss of IGF-I activity may not be uncommon in patients born small for gestational age. The characterization of this complex phenotype and identification of such molecular defects have therapeutic implications, particularly now that, in addition to GH, recombinant IGF-I is available for clinical use.

Antimicrobial activity of Enterococcus faecium ef 55 against Salmonella enteritidis in chicks.

The protective effect of Enterococcus faecium EF 55 against Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was studied in 1-day-old chicks. The EF 55 strain (isolated and characterised by the authors earlier) was applied daily (1.10(9) CFU/0.2 ml PBS) for 7 days. Oral inoculation of the SE PT4 strain was performed on day 8 in a single dose of 5.10(8) CFU/0.2 ml PBS. The experiment lasted for 21 days. Samples were collected on day 1 of the experiment to verify the absence of Salmonella, on day 8 to check colonisation of EF 55 and immunological status in experimental birds, and on days 2, 4, 6, 8 and 14 after SE PT4 infection of chicks. Strain EF 55 sufficiently colonised the digestive tract of chicks after 7 days of application. The highest numbers of EF 55 in the faeces of chicks were observed before SE infection and persisted to day 6 post infection (p.i.) in both the EF and EF+SE groups. PCR confirmed the identity of the EF 55 strain. The counts of SE PT4 strain in faeces of the EF+SE group were significantly reduced in comparison to those in the SE group on days 2 and 14 p.i. (P < 0.01). The significant reduction of salmonellae in the caecum was recorded at the end of the experiment (day 14 p.i.) in the EF+SE group in comparison to the SE group (P < 0.01). At day 4 p.i., colonies of S. Enteritidis PT4 were found in the liver of chicks of the SE group in a higher concentration than in chicks of the EF+SE group (P < 0.001). Salmonellae were isolated from the liver until days 8 and 6 p.i. in the SE and EF+SE groups, respectively. The mean values of actual lymphocyte subpopulations in the blood and the relative percentage of caecal intraepithelial lymphocyte subpopulations (CD4, CD8, CD44, TCR, MHC II and IgM) were not influenced at a statistically significant level by the application of the EF 55 and/or the SE PT4 strain. The results demonstrate the antimicrobial effect of E. faecium EF 55 against S. Enteritidis PT4.

Effect on the immune system of germ-free piglets of probiotics potentiated with polyunsaturated fatty acids.

The present study investigated the influence of polyunsaturated fatty acids (PUFA) on the immune system of germ-free piglets. Oil with increased content of omega-3 PUFA was administered to piglets from the experimental group (EG) for four weeks. Piglets from the control group (CG) received identical volumes of saline solution. At the age of 21 days both groups of germ-free piglets were inoculated perorally with Lactobacillus casei subsp. casei at a dose of 2 ml (1x10(8) mli). At the age of 28 days, i.e. after one-week colonisation of germ-free piglets with Lactobacillus casei subsp. casei, significant differences were recorded in phagocytic activity of neutrophils (PANe) and phagocytic activity of potentially phagocytizing cells (PA) (P < 0.05). Between EG and CG there have been observed no significant differences in absolute numbers of CD4+ and CD8+ T lymphocytes and numbers of IgM cells and in additional investigated parameters - number of CD2+ T lymphocytes, index of phagocytic activity of neutrophils (IPANe) and index of phagocytic activity (IPA). The total number of leukocytes (Le) in EG was also higher. Of the parameters determined in blood serum we observed a significant increase in concentration of alpha linolenic, eicosapentaenoic and docosahexaenoic acids and a parallel decrease in the level of arachidonic acid.

Differences in the development of the small intestine between gnotobiotic and conventionally bred piglets.

The effects of age, weaning and breeding conditions on the small intestinal morphology and the distribution of immunocompetent cells were investigated. The villus height and numbers of CD3+ T-lymphocytes, measured in the duodenum, jejunum and ileum, were determined in both the gnotobiotic and conventionally bred piglets. The diet of gnotobiotic piglets was composed of milk-replacement and feed mixtures. The application of milk replacement was finished on day 28. Conventional piglets were fed on sow's milk and feed mixtures. The animals were weaned on day 28. Small intestines were collected from 12 conventional and 12 gnotobiotic piglets slaughtered at the age of 2, 7, 14, 21, 28 and 35 days. The morphology results demonstrated that duodenal and ileal villi were significantly higher (p < 0.001) in gnotobiotic piglets during the entire period of the experiment. However, the weight of conventional piglets was higher during the experiment, in some cases significantly (p < 0.05). A marked reduction, in some cases significant (p < 0.001, p < 0.01 respectively), of the villi height on day 7 after the diet change for both groups of animals was recorded. The results demonstrate the differences in the gut development between both groups and their relationship to the breeding conditions. Nevertheless, the effect of crucial diet changes was observed independently of them. The immunohistochemistry results showed significantly (p < 0.001, p < 0.01 respectively) higher numbers of CD3+ T-lymphocytes in the jejunal villi of conventionally bred piglets. Similar results, in some cases significant (p < 0.001, p < 0.01 respectively), were also obtained from the other parts of the small intestine. These observations confirm reduced microorganism exposure under the gnotobiotic conditions.

The influence of omega-3 polyunsaturated fatty acids (omega-3 pufa) on lactobacilli adhesion to the intestinal mucosa and on immunity in gnotobiotic piglets.

The effect of application of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on intestinal colonization by Lactobacillus paracasei and on cellular immunity has been investigated in gnotobiotic pigs. The administration of polyunsaturated fatty acids positively affected the adhesion of Lactobacillus paracasei to the jejunal mucosa of gnotobiotic piglets. When compared to the control group, the number of Lactobacillus paracasei adhering to the jejunal mucosa was by 12% higher in piglets of the experimental group (5.10 log 10/cm2 vs. 4.55 log 10/cm2). The respective counts of Lactobacillus paracasei adhering to the ileal and colonic mucosa of 28 day old gnotobiotic piglets reached 4.45 and 5.05 log 10/cm2 in group C and 4.44 and 4.95 log 10/cm2 in group E. Omega-3 PUFA supplementation increased the phagocytic activity of neutrophils by almost 100% on day 28 of life as well as the subpopulations of lymphocytes (CD8) in the peripheral blood of germ-free piglets on day 21 of life. Our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA. The stimulatory effect of PUFA upon adhesion of lactobacilli could be used for enhancing the effectiveness of probiotics in inhibiting digestive tract pathogens.