N/A, thesis is written in chapters. / Thesis / Master of Science (MSc)

This study investigated effects of diet and probiotic supplement on stress in Thoroughbred foals at weaning. Twenty foals, whose dams were paired by age and breeding date, then randomly assigned to one of two diets prior to parturition, were used. Two groups were maintained on mixed grass pastures and fed supplements, one high in sugar and starch (SS) and one high in fat and fiber (FF) that met or exceeded NRC requirements. Half the foals on each diet were fed a commercial probiotic (Probios) containing lactic acid bacteria, while the rest were given a placebo. Plasma, fecal samples and behavioral ethograms were collected for four days pre- and post-weaning, and an ACTH response test was administered 48 h post-weaning. Cortisol, lactate, IgG, IgA and a-tocopherol were analyzed in plasma, volatile fatty acids and pH in feces. Foals fed FF had higher concentrations of IgA (P = 0.006), IgG (P = 0.012) and a-tocopherol (P = 0.005). Butyric and valeric acid concentrations were higher in feces of SS but not FF foals (P = 0.052), which may reflect better adaptation to forage in FF foals. Foals supplemented with probiotic had higher fecal lactate (P = 0.002) and lower fecal acetate (P = 0.0003) concentrations, suggesting that the lactic acid bacteria survived to the hindgut. Probiotic supplementation did not appear to benefit foals at weaning. Supplementation with FF may improve immune status and encourage a more diverse intestinal microbial population, enabling foals to better cope with the physiological stresses of weaning. / Master of Science

Cardiovascular disease (CVD) is the leading cause of death in the United States. Recently, the gut microbiota has been implicated in the pathophysiology and progression of CVD. Experimental evidence suggests that high fat feeding alters the functional composition of the gut microbiota (dysbiosis); leading to increased translocation of the pro-inflammatory, endotoxin, and increased production of the pro-atherogenic, trimethylamine-N-oxide (TMAO). Together, these changes are hypothesized to accelerate CVD progression. Conversely, administration of gut microbiota modulating agents, such as antibiotics and probiotics, attenuate high fat feeding induced CVD in rodent models. In humans, the capacity to produce TMAO following L-carnitine or phosphatidylcholine challenges is abolished after receiving broad spectrum antibiotics for a period of one week. However, whether gut modulation over a longer period of time decreases fasting serum endotoxin, fasting plasma TMAO, and CVD risk in response to high fat feeding has been unexplored in humans. To address these issues we conducted a randomized, placebo controlled, parallel group designed, controlled feeding study in healthy, non-obese males receiving the multi-strain probiotic, VSL #3 (or placebo), while a consuming a high fat diet for 4-weeks. First, we tested the hypothesis that VSL #3 would attenuate the rise in serum endotoxin and consequent arterial stiffening following high fat feeding in healthy, non-obese males. Second, we tested the hypothesis that VSL #3 would attenuate the rise in plasma TMAO concentrations following high fat feeding in healthy, non-obese males. In contrast to our first hypotheses, serum endotoxin concentrations and arterial stiffness did not change in response to high fat feeding or with VSL#3 treatment. Interestingly, VSL #3 significantly attenuated the increase in body mass (+ 1.4±0.4 vs. +2.3±0.3 kg; P < 0.05) and fat mass (+0.7±0.1 vs. + 1.4±0.3 kg; P < 0.05) following high fat feeding compared to the placebo. In contrast to our second hypothesis, probiotic supplementation did not attenuate the rise in plasma TMAO following high fat feeding. Future studies are necessary to elucidate the mechanisms responsible for the prevention of body mass and fat mass gain with VSL#3 supplementation following high fat feeding. In addition, studies are needed to determine whether higher doses of VSL #3, other single or multispecies probiotics, prebiotics, or synbiotics attenuate the production of the proatherogenic, TMAO. / Ph. D.

Probiotics are live nonpathogenic microorganisms capable of positively impacting the host by improving the natural gut microbial balance and promoting animal health. In ovo technology represents one means to administer probiotics and promote early colonization of beneficial bacteria for stimulating intestinal and immune system development, and warding off enteric threats. Three studies were conducted to evaluate the effects of in ovo administration of Primalac in broilers. The objective of the first study was to determine the effects of administering probiotics in ovo on hatchability, post-hatch performance and expression of immune-related genes in the ileum and cecal tonsils. On embryonic day 18, 360 eggs were injected with water, 1x105, 1x106, or 1x107 probiotic bacteria. Another 90 eggs remained uninjected as a negative control group. Measurements and tissue samples were taken on day of hatch (DOH) and days (d) 4, 6, 8, 15, and 22. A subsequent study was conducted to evaluate the effects of administering Primalac in ovo and in the diet on broiler chick hatchability, post-hatch performance, immune organ weights and ileal immune-related gene expression. At embryonic day 18, 1977 eggs were either not injected (negative control), dry-punched, or injected with 1x106 or 1x107 probiotic bacteria. Performance parameters were measured on DOH and d4, 6, 8, 14, 19, and 42, while immune organ weights and tissue samples were taken on DOH and d4, 6, 8, 14, and 20. A third study investigated the effects of in ovo administration of Primalac on hatchability, performance, immune organ weights, and lesion scores in broiler chicks exposed to Eimeria sp. At embryonic day 18, 210 eggs were injected with either sterile water or 1x106 probiotic bacteria. On d3 post-hatch, half of the chicks from each treatment group were challenged with a mixed inoculum of Eimeria acervulina, E. maxima, and E. tenella. Measurements and tissue samples were taken on DOH and d3, 9 and 15. The results of these studies suggest that in ovo Primalac supplementation does not negatively impact hatchability, enhances performance, modulates intestinal gene expression, and provides protection against a mixed Eimeria infection. / Ph. D.

Probiotic bacteria have been investigated in the prevention and treatment of various diseases and allergies. The current study was undertaken to determine the effect of eight probiotic Lactobacillus species against bacterial human skin pathogens using several techniques. Antimicrobial activity of lactobacilli against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes was evaluated using lactobacilli broth cultures (BCB) and cell free supernatant (CFS). Antimicrobial activity was significantly greater with BCB compared with CFS especially for Lactobacillus plantarum and Lactobacillus acidophilus. Lactobacilli and pathogen aggregation, biofilm formation and adhesion to keratin were assessed. L. casei and L. plantarum were selected for further study as they showed the greatest co-aggregation (18.02 ± 1.34% with L. casei and 14.92 ± 1.45% with L. plantarum) with the pathogens (16.63 ± 1.65% with S. aureus 3761 and 14.58 ± 1.68% with P. aeruginosa) and prevention of biofilm formation by the pathogens. The antimicrobial activity of human beta defensin-2 (hBD-2) alone or with L. plantarum against pathogens was assessed. The results with hBD-2 showed that hBD-2 (10 μg / ml for 5 h) and L. plantarum together were significantly more inhibitory against S. aureus than hBD-2 alone. The presence of NaCl reduced the effectiveness of hBD-2 alone and with L. plantarum. In the presence of L. plantarum, inactivation of mprF and dlt genes led to increased binding of hBD-2 by the bacterial cell wall, and then inhibition growth of bacterial cell wall. Studies investigated the effect of exposure of methicillin resistant Staphylococcus aureus (MRSA) to the supernatant of L. plantarum the susceptibility of MRSA to β-lactams. MRSA became sensitive to β-lactams when treated with culture supernatant of L. plantarum. Gene expression studies demonstrated that the mecR1-mecI-mecA-PBP2 signalling pathway was impeded by exposure to culture supernatant of L. plantarum and β-lactams. The studies reported here demonstrate a possible alternative approach to dealing with skin pathogens, which may have clinical implications especially with regard to MRSA infections, and continued research is advised.

Salmonellosis is a foodborne disease, poultry and poultry products are the main source of the disease. Many countries including Iraq still use antibiotics to control Salmonella. The increase in antibiotic resistant bacteria putatively caused by the overuse of antibiotics in agriculture has provided the incentive to look for alterna-tives to antibiotic to control diseases in livestock. Lactic acid bacteria with probiotic characteristics have the potential to beneficially affect the gut microflora of chick-ens and help reduce the incidence of Salmonella infection. In this study, Lactobacillus plantarum NCIMB 41607 was examined in vitro for in-hibitory effects against Salmonella and in vivo for its effects on the gut microflora and on the incidence of Salmonella infection. In separate studies Lb. plantarum was administered to chicks as fermented moist feed (containing 175 mmol/L lactic acid and 1x109 CFU/g of Lb. plantarum), in drinking water or applied to dry feed. Rifampicin resistance was used as a biomarker for the Lb. plantarum NCIMB 41607. Traditional and molecular microbiology including DGGE, RISA, and FISH were used with electron microscopy to assess the effect of Lb. plantarum on bac-terial population in the digestive system of chicks. Lactobacillus plantarum (NCIMB 41607) was found to reduce the growth of Salmonella Typhimurium and Salmonella Enteritidis by 4 Log CFU/g in a chicken simulated digestive system in vitro. The presence of rifampicine resistant Lactobacillus plantarum in the gut was con-firmed by replica plating and fluorescent in situ hybridisation with a strain specific probe. In general, the bacterial population in the guts of chicks fed Lactobacillus plantarum was more diverse in the guts of chicks fed control feed. There was no significant effect on Salmonella infection in Ross chicks compared with control. However, with specific pathogen free chicks the Salmonella infection was reduced. The intestinal villi lengths in FMF group were increased. These studies demonstrate that probiotics can affect the microflora in the chicken gut. Lactobacillus plantarum NCIMB 41607 may have the potential to control of Salmonella Typhimurium infection in chicken and continued research is advised.

Genes, pathways & transcription factors involved in probiotic mediated resolution of gut inflammation in IL10-KO mice an animal model of Inflammatory Bowel Disease. An integrated Gene, Protein and Bioinformatics Approach The IL10-KO mouse is a model of human inflammatory bowel disease (IBD), used to study host microbial interactions and potential therapeutics. Affymetrix microarray and proteomics analysis on colon of WT and IL10-KO mice and cecum of IL10-KO and WT mice orally administered with and without probiotic VSL#3 was performed and identified signalling pathways and transcription factors relevant to gut inflammation and anti-inflammatory probiotics. Results were validated by Real-time PCR, immunocytochemistry, proteomics, histopathology and via pathway signature analysis of publicly available microarray data. Changes in metabolically active bacteria in response to VSL#3 were assessed with DGGE. Inflammation in IL10-KO mice was characterised by up-regulation of immune/inflammatory and down-regulation of lipid/xenobiotic metabolism and PPAR signalling. VSL#3 resolved inflammation in the cecum inducing down-regulation of genes in immune/inflammatory pathways, decrease in the number of CCL5 positive T cells and up-regulation of galectin2, known to trigger apoptosis of T cells. VSL#3 induced up-regulation of PPARα/PPAR signalling and lipid/xenobiotic metabolism, antagonistic to NFB signalling and reduced metabolically active bifidobacteria. Analysis of publicly available data showed results were relevant to human IBD, indicated that antigen processing/presentation is up-regulated early on during development of colitis in IL10-KO mice, identified the potential of PPARα/PPAR signalling to induce down-regulation of CCL5, CD3 & antigen processing/presentation, and the potential of the xenobiotic metabolism to induce down-regulation of cytokine-cytokine interaction & mitosis. As VSL#3 treatment of IL10-KO mice induced up-regulation of PPARα/PPAR signalling and xenobiotic metabolism these results provide a possible mechanistic explanation for the VSL#3 induced down-regulation of CCL5, CD3, antigen processing/presentation, cytokine-cytokine interaction and mitosis in the cecum of VSL#3 treated IL10-KO mice.

Inflammation has modulatory effects on the brain, particularly during development. These plastic changes can hold severe functional consequences. Perinatal hypoxia-ischemia (HI)-induced inflammation can result in cerebral palsy and cognitive impairment. In an attempt to reduce inflammation in the brain, we assessed the probiotic Bifidobacterium (B.) infantis as an HI intervention, using a rat model. Rat pups, developmentally equivalent to preterm infants, were exposed to chronic hypoxia from postnatal (PND) 3 –PND 10. Inflammation was assessed through hippocampal concentrations of the cytokine interleukin-6 (IL-6). Tissue was collected from pups on PND 10 and analyzed via enzyme-linked immunosorbent assay (ELISA). Results showed lower IL-6 concentrations in hypoxic groups , regardless of B. infantis administration. Qualitative observations suggested poor gut health in association with hypoxia and probiotic exposure. These preliminary findings support the chronic hypoxia exposure model of HI and suggest the association with IL-6 and HI events is less straightforward than expected. / October 2016

A procura por suplementos alimentares cresce a cada ano, pois o seu uso contínuo promove a melhora e manutenção da qualidade de vida do hospedeiro. Sustentado neste princípio, a ingestão de produtos probióticos, principalmente leites fermentados, passa a ser uma alternativa neste segmento do mercado. Os probióticos são suplementos alimentares que contem microrganismos vivos que trazem benefícios à saúde do consumidor pela manutenção e melhora do balanço microbiano do trato gastrintestinal. Existem vários gêneros de microrganismos que apresentam propriedades probióticas, destacando-se o gênero Lactobacillus; que exercem várias funções benéficas ao hospedeiro como a diminuição dos níveis de colesterol sangüíneo, efeito anticarcinogênico, adesão ao epitélio intestinal, melhora do sistema imune e a exclusão competitiva, conhecida também como efeito barreira. Dentro deste contexto, o presente trabalho teve como objetivo avaliar o efeito de inibição exercido por cinco cepas de Lactobacillus isoladas de fezes humanas: denominadas L. plantrum (Lac-01), L. plantarum (Lac-02), L. fermentum (Lac-03), L. fermentum (Lac-04) e L. fermentum (Lac-05) sobre o crescimento de E. coli O157:H7, Listeria spp., P. aeruginosa ATCC 27853, Salmonella typhi e Shigella spp , por meio da técnica de co-cultura. Os resultados revelaram que as cepas Lac-01, Lac-02 e Lac-03 foram capazes de inibir, em diferentes níveis, o crescimento das cepas patogênicas avaliadas, sendo este efeito mais acentuado para S. typhi e Shigella spp. Observou-se ainda que a cepa Lac-04 não apresentou efeito de inibição sobre E. coli O157:H7 e P. aeruginosa, ao passo que a cepa Lac-05 inibiu o crescimento de P. aeruginosa, S. typhi e Shigella spp., sendo incapaz de inibir E. coli O157:H7 e Listeria spp, nas condições estudadas. Posteriormente ao se verificar a produção de substâncias antimicrobianas por meio do método \"spot-test\", observou-se o efeito positivo de inibição exercida pelas cepas de Lactobacillus sobre o crescimento dos respectivos patógenos. Verificou-se também, por meio de testes enzimáticos específicos, que as substâncias antimicrobianas produzidas pelas cepas de Lactobacillus não foram sensíveis às enzimas utilizadas, não podendo afirmar, desta forma, que estas substâncias são bacteriocinas. Os testes de co-agregação demonstraram que as cepas Lac-04 e Lac-05 exerceram melhor efeito sobre os patógenos, quando comparadas com as cepas Lac-01 e Lac-02. A cepa Lac-03 não exerceu efeito de coagregação com nenhum dos patógenos avaliados. Os testes de autoagregação revelaram que as cepas Lac-01, Lac-02, Lac-04 e Lac-05 exerceram efeito de autoagregação acentuado. Observou-se ainda que nenhum dos patógenos estudados foi capaz de autoagregar. / The probiotics are special food that contain live microorganisms that promote beneficits to consumer health through maintenance and improvement of microbial balance of gut tract. There are many microorganisms species used in probiotics products, standing out Lactobacillus species that produce many beneficies to consumer like decrease of blood cholesterol level, anti-carcinogenic effect, adhrence to intestinal epithelium, immune system stimulation and the competitive exclusion, also known as barrier effect. In this regard, the present work aimed to evaluate the inhibition effect exerted by five Lactobacillus strains, isolated from humam faeces: denominate, L. plantarum Lac-01, L. plantarum Lac-02, L. fermentum Lac-03, L. fermentum Lac-04 e L. fermentum Lac-05 on the gowth of E. coli O157:H7, Listeria spp., Pseudomonas aeruginosa ATCC 27853, Salmonella typhi and Shigella spp., by means of coculture technique. The results showed that the strains Lac-01, Lac-02 and Lac-03, were able to inhibit, at different levels, the growth of pathogenic strains, and this effect was more accentuated on S. typhi and Shigella spp. It was also observed that the strain Lac-04 did not present inhibition effect on E. coli O157:H7 and P. aeruginosa ATCC 27853, and the Lac-05 inhibited the growth of P. aeruginosa, S. tyhpi and Shigella spp., and showed no effect on E. coli O157:H7 and Listeria spp. Afterwards, the production of antimicrobial substance was verified by means of \"spot-test\" method, showing the positive inhibition effect exerted by all Lactobacillus strains on the growth of the respective pathogens. By means of specific enzymatic test, the antimicrobial substances produced by lactobacillus strains were not sensible to the action of enzymes proteinase-K, papain and pepsin, but this is not enough to affirm that this substances are bacteriocins. The coaggregation test demonstrated that the strains Lac-04 and Lac-05 exerted better effect on the pathogens, when compared to strains Lac-01 and Lac-02. The strain Lac-03 did not coaggregate with any pathogenic strains. The autoaggregation test showed that the strain Lac-01, Lac-02, Lac-04 and Lac-05 exerted accentuated auto-aggregation effect. The studied pathogen was not able to autoaggregate.

Orientador: Raphael Lucio Andreatti Filho / Banca: Paulo Lourenço da Silva / Banca: Renée Laufer Amorim / Resumo: O objetivo deste trabalho foi verificar por meio de imunoistoquímica se a inoculação oral de culturas de Lactobacillus acidophillus, L. reuteri, L. salivarius e a microbiota cecal de aves estimula a resposta imune celular no intestino de pintos de linhagem comercial, desafiadas ou não por Salmonella Enteritidis, na forma de infiltrado leucocitário por células T CD3+, CD4+ e CD8+. Foram utilizados 320 aves com um dia de vida, não sexados e livres de quimioterápicos. Cada grupo foi subdividido em quatro subgrupos com 20 aves cada e classificados em grupo que não recebeu tratamento (controle negativo), tratamento com L. spp ou MC, tratamento com L. spp ou MC e desafio com S. Enteritidis e tratamento com S. Enteritidis (controle positivo). Os resultados obtidos mostram que o tratamento com L. reuteri, L. salivarius, L. acidophillus ou microbiota cecal e o desafio com S. Enteritidis, determina resposta imune na forma de infiltrado leucocitário por linfócitos CD3+, CD4+ e CD8+ no epitélio e na lâmina própria do duodeno, jejuno e ceco de aves com até 12 dias de idade. / Abstract: The bacteria of the Lactobacillus sort have been very studied currently had to its important paper in the process of competitive exclusion where they contribute in the reduction of pathogenically genus, in special infections for Salmonella spp. it saw oral in the first days of life. These bacteria with probiotically characteristics provide beneficial effect to the general health of the host, through the stimulation and anti tumor modulation of the immune reply and properties, had probably the action on the cellular immune system. The objective of this work was to verify if Lactobacillus acidophillus, L. reuteri, L. salivarius and microbiota cecal of broiler chicken have been imunostimulated activities through its inoculation in birds of commercial ancestry with one day of life, defied or not for Salmonella Enteritidis, determining immune reply in the form of infiltrated leucocytes for cells T in the epithelium and intestinal proper blade.The gotten results demonstrate that the cultures of L. acidophillus, L. reuteri, L. salivarius and microbiota cecal defy or not with S. Enteritidis, they determine immune reply on the part of the host in the infiltrated form of leucocytes for lymphocytes CD3+, CD4+ and CD8+. / Mestre