The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Thesis (Ph.D.). / Written for the School of Dietetics and Human Nutrition. Title from title page of PDF (viewed 2007/08/29). Includes bibliographical references.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on Sept. 21, 2009). Thesis advisor: Dr. Azlin Mustapha. Includes bibliographical references.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on Oct. 6, 2009). Thesis advisor: Dr. Azlin Mustapha. Includes bibliographical references.

With obesity and type 2 diabetes on the rise, research is trying to find ways to reverse or slow its progress. Soy diets have been shown to be effective in doing so but have variable results. One variable that may affect soy's effectiveness is intestinal microflora. This experiment used female Zucker Diabetic Fatty (ZDF) rats that develop type 2 diabetes when fed high-fat diet and is similar to that of human development of type 2 diabetes. This study used soy diets designed to modify intestinal bacteria with probiotics or prebiotics: control, 2.5% fructooligosaccharide (FOS), 2.5% B. lactis, or 2.5% L. acidophilus. Food intake, body weight, and glucose levels were evaluated weekly throughout the study. At the end of a 23 day period total body lipids were assessed, as well as, glucose levels. The percent body lipids in the B. lactis group were higher than all other groups (p>0.05). The B. lactis and L. acidophilus groups had seemingly higher glucose levels; however, the statistical analysis was insignificant due to high variation between groups. Urine samples showed B. lactis and L. acidophilus groups had three rats with glucose levels of 500 mg/dl or above while control and FOS groups had one rat each in this category. This study showed no improvement to obesity and diabetic parameters through the microflora modifications used. In fact, some parameters worsened indicating a need for continuing research of soy with intestinal microflora modification.

The equine cecal microbiome is an incredibly diverse ecosystem that is critical to the overall health of the horse. It is of particular interest to equine researchers because of the link between colic and the bacterial profile residing within the cecum. We investigated ten probiotics for their ability to reduce numbers of previously identified pathogenic microorganisms: Streptococcus bovis/equinus complex (SBEC), Escherichia coli K-12, Escherichia coli general, Clostridium difficile, and Clostridium perfringens. A preliminary in vitro study was used to measure the reduction in opportunistic bacteria that are commonly found in the equine gastrointestinal tract. The second in vitro was designed as a titration study using the three most effective probiotics from the first project. In this second stage of testing, different dosage levels were utilized to determine if dosage had an effect on bacterial reduction potential. Dosage levels included manufacturer’s recommended dosage (1x), twice the recommended dosage (2x), and three times the recommended dosage (3x). Lastly, an in vivo study was conducted using three cecally-cannulated horses in a Latin square design in order to measure opportunistic bacteria reduction potential in the live horse model. Cecal fluid characteristics, pH, volatile fatty acids, and ammonia concentrations were measured, along with bacterial concentrations. In the initial in vitro experiment, we observed that all probiotic treatments numerically decreased the bacterial concentrations in comparison to the control. There were three products that decreased bacterial concentrations most consistently: Command FT, CRS, and SmartDigest Ultra. These probiotics were chosen for a titration in vitro study. Selected probiotics were dosed at the recommended dose (1x), as well as at two times the recommended dose (2x) and three times the recommended dose. In the titration in vitro experiment, SmartDigest Ultra increased (P < 0.01) bacterial concentrations of E. coli K12 and C. perfringens at 2x and 3x. Due to a lack of statistical significance for Command FT and CRS (P ≥ 0.10), cost efficiency was used as a selection criteria. Command FT was approximately $0.27 per dose and CRS was approximately $0.18 per dose. Therefore, CRS was chosen for further testing in the following in vivo experiment. In the in vivo experiment, CRS was dosed at one times (1x) and two times (2x) the recommended dose. We observed that blood parameters, cecal fluid characteristics, and bacterial concentrations were not statistically (P ≥ 0.10) altered by treatment. Numerical increases for bacterial concentrations were observed for SBEC at 1x and 2x, C. difficile at 2x, and C. perfringens at 2x. Numerical decreases were observed for E. coli K12 at 1x and 2x and E. coli general at 1x and 2x. Overall, this study suggests that the selected probiotics can be safely used at the recommended dose.

Made available in DSpace on 2016-04-01T17:54:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-10. Added 1 bitstream(s) on 2016-04-01T18:00:37Z : No. of bitstreams: 1 000860697.pdf: 1733916 bytes, checksum: 319f98dc74fda5996433b386d24f96c0 (MD5) / A alta incidência de candidoses causadas por Candida albicans e a capacidade de adaptação desta espécie, assim como resistência aos antifúngicos impulsionam o desenvolvimento de pesquisas com terapias alternativas para controle dessa infecção. O objetivo desse trabalho foi avaliar a influência de Lactobacillus rhamnosus e produtos do seu metabolismo contra C. albicans, avaliando-se a patogenicidade e a expressão de genes que regulam a formação do biofilme de C. albicans, in vitro e in vivo em modelo invertebrado de Galleria mellonella. Foram utilizadas cepas de C. albicans ATCC 18804 e L. rhamnosus ATCC 9595 (provenientes do Laboratório de Microbiologia do Instituto de Ciências e Tecnologia de São José dos Campos / UNESP). Para o estudo foram preparadas duas suspensões de L. rhamnosus, uma contendo a porção celular e a outra com o sobrenadante da cultura, livre de células. O estudo da patogenicidade in vitro de C. albicans foi avaliado pelos testes de formação de biofilme e quantificação de UFC/mL de C. albicans, análise da atividade metabólica pelo método do XTT e filamentação de C. albicans. A análise da curva de sobrevivência de Galleria mellonella foi utilizada para a avaliação in vivo da influência de L. rhamnosus na infecção causada por C. albicans. Os genes, BCR1, EFG1, CPH1, HWP1, ALS1 e ALS3 de C. albicans foram quantificados pela RT-PCR após a interação em modelo de biofilme. No estudo in vitro L. rhamnosus foi capaz de inibir a formação do biofilme, filamentação e reduzir a atividade metabólica de C. albicans. Esses efeitos também foram observados quando utilizado a suspensão do sobrenadante da cultura de L. rhamnosus, apontando possível produção de substâncias com efeito inibitório. Os resultados obtidos in vivo apontam que L. rhamnosus e seu sobrenadante protegeram G. mellonella da infecção por C. albicans, porém a suspensão do sobrenadante apresentou melhores resultados, visto.... / The high incidence of candidiasis caused by Candida albicans and the adaptability of this species, as well as resistance to antifungal drive the development of research on alternative therapies to control this infection. The aim of this study was to evaluate the influence of Lactobacillus rhamnosus and products of their metabolism against C. albicans, evaluating the pathogenicity and the expression of genes that regulate the formation of C. albicans biofilms in vitro and in vivo in invertebrate model Galleria mellonella. Strains of C. albicans ATCC 18804 and L. rhamnosus ATCC 9595 were used (from the Microbiology Laboratory of the Institute of Science and Technology of São José dos Campos / UNESP). For the study were prepared from L. rhamnosus two suspensions, one containing the serving cell and the other with the culture supernatant free cells. The study of in vitro pathogenicity of C. albicans was assessed by biofilm formation testing and quantitation of CFU / ml of C. albicans, analysis of metabolic activity by XTT method and C. albicans filamentation. The analysis of the Galleria mellonella survival curve was used to evaluate in vivo the influence of L. rhamnosus in infection caused by C. albicans. Genes, BCR1, EFG1, CPH1, HWP1, ALS1 and ALS3 of C. albicans were quantified by RT-PCR after interacting in biofilm model. In vitro evaluation L. rhamnosus was able to inhibit biofilm formation, filamentation and reduce the metabolic activity of C. albicans. These effects were also observed when using the suspension culture supernatant of L. rhamnosus, indicating the possible of production substances with inhibiting effect. The results obtained in vivo indicate that L. rhamnosus and its supernatant protected G. mellonella of C. albicans infection, but the suspension of the supernatant showed better results, as it increased the survival caterpillar G. mellonella. The results of gene expression have shown that ....

Made available in DSpace on 2014-06-11T19:33:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-22Bitstream added on 2014-06-13T21:05:41Z : No. of bitstreams: 1 lodi_cs_dr_araca.pdf: 472874 bytes, checksum: c9c4033eb4b377f2e55af453639c1621 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um número crescente de produtos contendo probióticos está disponível no mercado e vem sendo utilizados pelos consumidores. Diante disso, o objetivo deste trabalho foi avaliar in situ, in vivo e in vitro a relação da bactéria probiótica com a cárie dentária. No estudo in situ investigou-se a cariogenicidade do leite fermentado contendo probióticos através da quantificação dos açúcares totais e redutores presente no produto, análise do seu efeito na desmineralização do esmalte dental bovino, análise microbiológica da saliva antes e após o período experimental, análise microbiológica e quantificação dos carboidratos álcalis-solúveis presente no biofilme. Para isso, dez voluntários utilizaram um dispositivos contendo 4 blocos de esmalte dental bovino. O experimento consistiu de 3 etapas de 14 dias cada onde os voluntários gotejaram solução de sacarose 20% ou a solução de tratamento (Tratamento A - Yakult® ou Tratamento B - Batavito®) 8X/dia. Decorrido o período experimental, o biofilme e a saliva foram analisados quanto a quantidade de microrganismos totais (MT), Streptococcus totais (ST) e Streptococcus do grupo mutans (SM), Lactobacillus (L). Para os dados de dureza foram calculados a porcentagem de variação de dureza superficial e a perda integrada de dureza de subsuperfície. Após o tratamento B foi observado menor quantidade de MT no biofilme quando comparado com o tratamento A, mas não diferiu da solução de sacarose 20%. Na saliva, o tratamento com solução de sacarose 20% diminuiu a quantidade de MT e ST, e aumento a quantidade de SM. O tratamento A provocou uma diminuição na quantidade de MT, ST e SM e o tratamento B diminuiu a quantidade de MT. Para os dados... / Probiotics are live microorganisms, which when administered in adequate amounts, confer a health benefit on the host. An increasing number of probiotic-containing products are available, and these products have been orally consumed. However, the objective of this study was to evaluate in situ, in vivo and in vitro the relation between probiotic bacteria and dental caries. In the in situ and in vivo study it was evaluate the effect of 2 probiotic-containing fermented milk on biofilm and saliva microorganisms and on enamel surface. The in situ study was performed in 3 phases: 20% sucrose, treatment A (Yakult®) and treatment B (Batavito®). Salivary microorganisms were counted at baseline and after and biofilm was analyzed just after the trial period. In vivo study was performed in 2 phases: treatment C (Yakult®) and treatment D (Batavito®). The saliva was collected at baseline and at the end of the trial period for microbiological analysis. In the in situ study, biofilm data showed less total microorganisms (TM) after treatment B than treatment A but similar to 20% sucrose. In the saliva, 20% sucrose decreased TM and total streptococci (TS), and increased mutans streptococci (MS). Treatment A significantly decreased TM, TS and MS. Treatment B decreased TM. It was observed less MS in treatment B when the final data were compared among the treatments. Treatment B differed from the other treatments in relation to final microhardness, percentage change of surface hardness and integrated loss of subsurface hardness (p<0.05). In vivo study showed that just treatment D decreased all microorganisms. It was observed higher lactobacilli (L) in treatment D when the baseline data were compared among the groups. In the in vitro study, it was determined the ability of probiotic bacteria to prevent primary caries development... (Complete abstract click electronic access below)

Orientador: Antonio Olavo Cardoso Jorge / Banca: Juliana Campos Junqueira / Banca: Graziella Nuernberg Back Brito / Banca: Liliana Scorzoni / Banca: Fernanda Malagutti Tomé / Resumo: Devido ao aumento das infecções causadas por Candida albicans e espécies não-albicans, surge a necessidade de novas estratégias terapêuticas, como a identificação de cepas bacterianas com características probióticas. Assim, o objetivo foi avaliar a ação antimicrobiana de cepas clínicas de Lactobacillus paracasei, Lactobacillus fermentum e Lactobacillus rhamnosus sobre diferentes espécies de Candida (C. albicans, C. glabrata, C. krusei e C. tropicalis). Foram utilizadas cepas de Lactobacillus isoladas da cavidade bucal de indivíduos livres de cárie e cepas de Candida isoladas de lesões de candidose orofaríngea obtidas em estudos anteriores. Para estudo in vitro, foram formados biofilmes monoespécies de Candida (Grupo controle) e biofilmes mistos de Candida e Lactobacillus, nos quais os lactobacilos foram acrescentados antes da cepa de Candida (Grupo Profilático), após a cepa de Candida (Grupo Terapêutico) ou ao mesmo tempo da cepa de Candida (Grupo Simultâneo). Após 48 h de incubação, foi realizada a contagem do número de células viáveis de Candida (UFC/mL). Para estudo in vivo, os micro-organismos foram inoculados em larvas de G. mellonella e o desenvolvimento da candidose foi monitorado pela curva de sobrevivência, estudando-se os grupos experimentais descritos acima. Nos resultados in vitro, foi verificado que os efeitos inibitórios mais significativos de Lactobacillus sobre Candida ocorreram nos grupos profiláticos. As cepas de C. tropicalis e C. krusei foram mais sensívei... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract : Due to the increase of infections caused by Candida albicans and non-albicans species, there is a need for new therapeutic strategies, such as the identification of bacterial strains with probiotic characteristics. The objective of this study was to evaluate the antimicrobial action of Lactobacillus paracasei, Lactobacillus fermentum and Lactobacillus rhamnosus strains on different Candida species (C. albicans, C. glabrata, C. krusei and C. tropicalis). We used Lactobacillus strains isolated from the oral cavity of caries-free and Candida strains isolated from lesions of oropharyngeal candidiasis. For in vitro study, monospecies of Candida (Control Group) and mixed biofilms of Candida and Lactobacillus were prepared, in which the lactobacillus were attached before the Candida strain (Profile Group), after a Candida (Therapeutic Group) strain or same time as the Candida strain (Simultaneous Group). After 48 h of incubation, we counting the number of viable Candida cells (CFU/mL). For in vivo study, we inoculate the microorganisms in larvae of G. mellonella and the development of the application for monitoring by survival curve, studying the experimental groups described above. In the in vitro results, we find that the most significant inhibitory effects of Lactobacillus on Candida occurred in the prophylactic groups. The strains of C. tropicalis and C. krusei were more sensitive to the action of Lactobacillus than C. albicans and C. glabrata. In addition, it we verified that the three species of Lactobacillus studied (L. paracasei, L. rhamnosus and L. fermentum) had an inhibitory action on Candida. In relation to the in vivo study, we verified that the prophylactic groups had more inhibitory effects on an experimental candidose in relation to the therapeutic groups, leading to a significant increase in the survival rates of the larvae. In relation to the .....(Complete abstract click electronic access below) / Doutor