Tesis sobre el tema "Bacterial kidney disease (Fish disease)"

The role of reactive oxygen and nitrogen intermediates in the immune response of rainbow trout to R.s. was investigated. The early events occurring when the pathogen interacted with trout macrophages were assessed in terms of the respiratory burst elicited. Live R.s. elicited a respiratory burst, which was enhanced by heat-killed microorganism. This phenomenon, though, was not observed using UV-killed bacteria. Both responses were enhanced when a combination of LPS and TNF was used to activate the macrophages prior to contact R.s. Further studies demonstrated that both compounds synergised to enhance superoxide (O2) production, and that this was correlated with the ability to kill the pathogen. Opsonisation of R.s. with serum factors also increased the respiratory burst, but no difference was found between normal serum and heat-inactivated serum. The role of NO in the immune response of rainbow trout is also studied. Though no evidence of NO production was found in vitro, i.p. injection of live R.s. produced higher NO levels in serum as compared to controls. Fish injected with a virulent strain showed higher levels of NO than controls and than fish injected with an avirulent strain and other strains of unknown virulence. Fish vaccinated with killed R.s. and FIA also showed a significant increase in NO levels, but only four days after vaccination, decreasing thereafter, at both doses of vaccine tested. Injected of Brivax II, an attenuated strain of Aeromonas salmonicida, did not produce a significant increase of NO. RT-PCR was used to detect the expression of the iNOS in different tissues of rainbow trout. iNOS expression was seen only in gill and kidney after i.p. injection. iNOS was detected in the gills 6 h after injecting live R.s. and the expression was still present at day 5. iNOS was detected in the kidney 24 h after injection but was switched off at day 3. After bath challenge with the bacterium, iNOS was expressed in gill, gut and kidney, but the expression varied in each fish. No iNOS expression was found in macrophages isolated from challenged fish.

Pacific salmon reared commercially off of the Coast of British Columbia suffer great mortality losses to Bacterial Kidney Disease (BKD), caused by the diplobacillus bacterium Renibacterium salmqninarum. This thesis investigates the effects of environmental conditions on the growth performance and disease susceptibility of salmonids reared in captivity. I found that growth rate of chinook salmon was significantly higher in fish fed to 100 compared to 67 % of satiation during the first 175 days of saltwater rearing but not during the first winter. Feed coversion rate was significantly higher for fish fed at 100 % of satiation compared to 67 % of satiation and higher during the winter compared to summer and fall, irrespective of feeding level. Mortality rates were significantly higher during the summer than during the fall or winter, irrespective of experimental treatment. The last BKD sampling period (day 263) revealed that infection rates were directly proportional to stocking densities of 1.5 to 4 kg.m⁻₃. Hatchery-reared chinook salmon held in freshwater aquaria had significantly lower hematocrit and plasma cortisol concentration increases in response to increased stocking density than did their wild counterparts. Crowding of hatchery-reared and wild chinook salmon resulted in equally increased mortality rates for both groups of fish. Day 33 plasma cortisol concentrations in Atlantic salmon held at three stocking densities were directly proportional to stocking densities of 8 to 64 kg.m⁻₃. The ability of anterior kidney lymphocytes from these fish to produce antibody-producing cells was inversely proportional to the density at which the fish were held.
Land and Food Systems, Faculty of
Graduate

Renibacterium salmoninarum, the etiological agent of bacterial kidney disease (BKD) of salmonid fish, is a pathogen of great concern among fisheries and the aquaculture industry worldwide. Previous investigations have indicated the pathogenesis of BKD is complex. It is a chronic, multisystemic, granulomatous disease with a number of potential immunomodulatory effects on the host. Given the current limitations for treatment and control of BKD, it is imperative that the pursuit of development of methods of prevention, namely management strategies and vaccination, be continued. To do so, the immunology of BKD must be elucidated in order to better understand and manipulate the associated immune responses to our advantage. This dissertation is composed of four chapters which relate to BKD and the associated immune responses of three species of susceptible salmonid fish as follows: Exogenous stress factors, through stress-induced immunosuppression, have been shown to influence BKD development in cultured salmonids. Chapter 1 examines the effects of two environmental stressors common to fish culture, overcrowding and overfeeding, as they affect BKD development and R. salmoninarumantigen prevalence among juvenile chinook salmon (Oncorhynchus tshawytscha) Immunomodulatory interaction between pathogen and host in BKD is widely reported and merits further investigation. Particularly, the immunological parameters affected and the role of the extracellular protein (ECP) of R. salmoninarum are of interest. Chapter 2 examines the in vivo immune response of rainbow trout (Oncorhynchus mykiss) following exposure to the ECP in terms of both humoral and cell-mediated immunological parameters, including the immune response against another bacterial pathogen. Chapter 3 addresses the in vitro effects of the ECP upon specific splenic immunocyte functions, phagocytosis and respiratory burst activity, in brook trout (Salvelinus fontinalis). The immune-complex mediated hypersensitivity reported to occur with BKD has considerable ramifications for control measures involving immunostimulation via antigen exposure. Further investigation is warranted to discern the significance and consistency of immunological hypersensivity in BKD pathogenesis. Chapter 4 examines the renal lesions, including immunopathologic changes and indications of immune-mediated disease, of brook trout exposed to R. salmoninarum.
Ph. D.

A new medium, designated Ribose Ornithine Deoxycholate agar (ROD), was developed and used in field trials at two fish farms where fish were known to have ERM. The medium indicated that Yersinia ruckeri could occur in faeces four to six weeks before appearing in the kidney. Fxurther epidemiological studies dealing with fish condition, performance, disease signs and water isolation are presented. These factors are discussed in relation to husbandry and management practices. The minimum uihibitory concentration (MIC) values for 124 strains of F. ruckeri were detemuned. The effect of oxolinic acid, oxytetracycline and a potentiated sulphonamide, on growth patterns over a 72 h period, was also determined. Results showed the bactericidal or bacteriostatic nature of each antimicrobial agent. It was possible to increase the MIC for oxoUnic acid, oxytetracycline and a potentiated sulphonamide usmg an in vitro technique. Attempts to decrease resistance to oxolinic acid were unsuccessful. However, it was possible to reduce the MIC's for o^etracycline and a potentiated sulphonamide. The recovery of Y. ruckeri, after artificial challenge, was less from the faecal and kidney material of a vaccinated group of fish compared with a non-vaccinated group. An E L I S A technique indicated that although there was no detectable serum antibody there was a local mucosal response i n vaccinated fish. Uptake of F. ruckeri antigen was demonstrated in vaccinated and non-vaccinated fish using an immunoperoxidase technique. However, only vaccinated fish appeared to take up the antigen by an active process. A survey of salmonid farms showed that there was a tendency for those sites where ERM had been diagnosed, irrespective of vaccination, to be larger table farms with production ranging up to over 200 tonnes p:a. Certain environmental and husbandry factors were reported as coinciding with the appearance of F. ruckeri. A slight tendency to consider that vaccines had failed i n some way was also indicated. This and the use of antimicrobial agents are discussed.

Thesis (D.Sc.N.S.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1993 (Nutritional Sciences).
Includes bibliography (leaves 176-187)
This dissertation has been presented in two related studies: A. Fish Oil Reduces Proteinuria and Interstitial Injury but not GIomerulosclerosis in the Milan Nomotensive Rat Rats of the Milan Normotensive strain (MNS) spontaneously develop severe Proteinuria and excessive glomemlar thromboxane (Tx)A2 PrOduction at a young age. These are accompanied by podocyte alterations and progressive focal glomerulosclerosis (FGS) and interstitial fibrosis. Since previous studies showed that pharmacologic... [TRUNCATED]

Proliferative Kidney Disease (PKD) caused by the Malacosporean parasite Tetracapsuloides bryosalmonae, is presently the most economically damaging disease of British rainbow trout farming, costing the industry in excess of £2.5 million per annum in the UK alone. With no vaccine or prophylactic treatment available, and only management techniques currently adopted to minimise the stress and mortality associated with the disease, alternative approaches must now be considered. This document investigates if selective breeding for PKD resistance is possible by assessing the level of additive genetic variation, and calculating the subsequent estimates of heritability, for commercial strains of rainbow trout. During a PKD outbreak on a commercial farm, 1500 communally reared juvenile rainbow trout from two strains (Houghton Spring and Isle of Man) were sampled on a single day, their body weight and fork length measured, and severity of kidney swelling scored according to the scale of Clifton-Hadley et al. (1987). Fish were assigned to individual families using microsatellite parentage assignment. Significant additive genetic variation was observed in the population, and families were ranked according to estimated breeding values. A combined estimate of heritability (h2 = 0.19 ± 0.08) for kidney score suggests the population will respond well to selective breeding for kidney score, which may be deemed a measure of resistance, whilst the favourable genetic correlations between kidney score and the production traits measured suggest simultaneous selection for kidney score and growth traits should also be effective. In order to support the findings of the initial research, controlled challenge experiments were conducted. Using the family EBV information on kidney score from the IoM strain (due to its certification as a disease-free site), four females, two with high and two with low response to PKD, were each crossed with a randomly selected neomale to produce twenty two families for PKD challenge experiments. The PKD experimental challenges showed evidence of additive genetic variation to kidney score over an eleven week period, supporting initial findings. A low score was deemed as evidence of greater resistance to the parasite in this study. Although female EBV was taken into consideration in the statistical model, there was found to be no significant difference in resistance according to family. Immunohistochemistry stained kidney sections from each individual involved in the challenges proved kidney score correlated significantly to the number of parasites in the kidney, suggesting that the scale of Clifton-Hadley et al. (1987) is a sufficient and accurate basis on which to describe the severity of PKD, and infection level in rainbow trout. Having discovered evidence that furunculosis, causative agent Aeromonas salmonicida, plays a major role in the mortality of fish suffering from PKD in the field, the bacterial disease was investigated to assess the resistance of the same families used in the PKD challenges. Twenty one of the families were used to discover that additive genetic variation for resistance to furunculosis is apparent when assessed as both a binary and longitudinal trait, suggesting significant genetic improvement can be made to increase resistance to furunculosis in the IoM stock. No significant correlation was observed between kidney score, EBV, and resistance to this bacterium, but there was a positive phenotypic correlation found between furunculosis resistance and size, suggesting simultaneous selection for performance and resistance is possible within this population.

Protein-energy wasting (PEW) is commonly seen in patients with chronic kidney disease (CKD). The condition is characterised by chronic, systemic low-grade inflammation which affects nutritional status by a variety of mechanisms including reducing appetite and food intake and increasing muscle catabolism. PEW is linked with co-morbidities such as cardiovascular disease, and is associated with lower quality of life, increased hospitalisations and a 6-fold increase in risk of death1. Significant gender differences have been found in the severity and effects of several markers of PEW. There have been limited studies testing the ability of anti-inflammatory agents or nutritional interventions to reduce the effects of PEW in dialysis patients. This thesis makes a significant contribution to the understanding of PEW in dialysis patients. It advances understanding of measurement techniques for two of the key components, appetite and inflammation, and explores the effect of fish oil, an anti-inflammatory agent, on markers of PEW in dialysis patients. The first part of the thesis consists of two methodological studies conducted using baseline data. The first study aims to validate retrospective ratings of hunger, desire to eat and fullness on visual analog scales (VAS) (paper and pen and electronic) as a new method of measuring appetite in dialysis patients. The second methodological study aims to assess the ability of a variety of methods available in routine practice to detect the presence of inflammation. The second part of the thesis aims to explore the effect of 12 weeks supplementation with 2g per day of Eicosapentaenoic Acid (EPA), a longchain fatty acid found in fish oil, on markers of PEW. A combination of biomarkers and psychomarkers of appetite and inflammation are the main outcomes being explored, with nutritional status, dietary intake and quality of life included as secondary outcomes. A lead in phase of 3 months prior to baseline was used so that each person acts as their own historical control. The study also examines whether there are gender differences in response to the treatment. Being an exploratory study, an important part of the work is to test the feasibility of the intervention, thus the level of adherence and factors associated with adherence are also presented. The studies were conducted at the hemodialysis unit of the Wesley Hospital. Participants met the following criteria: adult, stage 5 CKD on hemodialysis for at least 3 months, not expected to receive a transplant or switch to another dialysis modality during the study, absence of intellectual impairment or mental illness impairing ability to follow instructions or complete the intervention. A range of intermediate, clinical and patient-centred outcome measures were collected at baseline and 12 weeks. Inflammation was measured using five biomarkers: c-reactive protein (CRP), interleukin-6 (IL6), intercellular adhesion molecule (sICAM-1), vascular cell adhesion molecule (sVCAM-1) and white cell count (WCC). Subjective appetite was measured using the first question from the Appetite and Dietary Assessment (ADAT) tool and VAS for measurements of hunger, desire to eat and fullness. A novel feature of the study was the assessment of the appetite peptides leptin, ghrelin and peptide YY as biomarkers of appetite. Nutritional status/inflammation was assessed using the Malnutrition-Inflammation Score (MIS) and the Patient-Generated Subjective Global Assessment (PG-SGA). Dietary intake was measured using 3-day records. Quality of life was measured using the Kidney Disease Quality of Life Short Form version 1.3 (KDQOL-SF™ v1.3 © RAND University), which combines the Short-Form 36 (SF36) with a kidney-disease specific module2. A smaller range of these variables was available for analysis during the control phase (CRP, ADAT, dietary intake and nutritional status). Statistical analysis was carried out using SPSS version 14 (SPSS Inc, Chicago IL, USA). Analysis of the first part of the thesis involved descriptive and bivariate statistics, as well as Bland-Altman plots to assess agreement between methods, and sensitivity analysis/ROC curves to test the ability of methods to predict the presence of inflammation. The unadjusted (paired ttests) and adjusted (linear mixed model) change over time is presented for the main outcome variables of inflammation and appetite. Results are shown for the whole group followed by analyses according to gender and adherence to treatment. Due to the exploratory nature of the study, trends and clinical significance were considered as important as statistical significance. Twenty-eight patients (mean age 61±17y, 50% male, dialysis vintage 19.5 (4- 101) months) underwent baseline assessment. Seven out of 28 patients (25%) reported sub-optimal appetite (self-reported as fair, poor or very poor) despite all being well nourished (100% SGA A). Using the VAS, ratings of hunger, but not desire to eat or fullness, were significantly (p<0.05) associated with a range of relevant clinical variables including age (r=-0.376), comorbidities (r=-0.380) nutritional status (PG-SGA score, r=-0.451), inflammatory markers (CRP r=-0.383; sICAM-1 r=-0.387) and seven domains of quality of life. Patients expressed a preference for the paper and pen method of administering VAS. None of the tools (appetite, MIS, PG-SGA, albumin or iron) showed an acceptable ability to detect patients who are inflamed. It is recommended that CRP should be tested more frequently as a matter of course rather than seeking alternative methods of measuring inflammation. 27 patients completed the 12 week intervention. 20 patients were considered adherent based on changes in % plasma EPA, which rose from 1.3 (0.94)% to 5.2 (1.1)%, p<0.001, in this group. The major barriers to adherence were forgetting to take the tablets as well as their size. At 12 weeks, inflammatory markers remained steady apart from the white cell count which decreased (7.6(2.5) vs 7.0(2.2) x109/L, p=0.058) and sVCAM-1 which increased (1685(654) vs 2249(925) ng/mL, p=0.001). Subjective appetite using VAS increased (51mm to 57mm, +12%) and there was a trend towards reduction in peptide YY (660(31) vs 600(30) pg/mL, p=0.078). There were some gender differences apparent, with the following adjusted change between baseline and week 12: CRP (males -3% vs females +17%, p=0.19), IL6 (males +17% vs females +48%, p=0.77), sICAM-1 (males -5% vs females +11%, p=0.07), sVCAM-1 (males +54% vs females +19%, p=0.08) and hunger ratings (males 20% vs females -5%, p=0.18). On balance, males experienced a maintainence or reduction in three inflammatory markers and an improvement in hunger ratings, and therefore appeared to have responded better to the intervention. Compared to those who didn’t adhere, adherent patients maintained weight (mean(SE) change: +0.5(1.6) vs - 0.8(1.2) kg, p=0.052) and fat-free mass (-0.1 (1.6) vs -1.8 (1.8) kg, p=0.045). There was no difference in change between the intervention and control phase for CRP, appetite, nutritional status or dietary intake. The thesis makes a significant contribution to the evidence base for understanding of PEW in dialysis patients. It has advanced knowledge of methods of assessing inflammation and appetite. Retrospective ratings of hunger on a VAS appear to be a valid method of assessing appetite although samples which include patients with very poor appetite are required to confirm this. Supplementation with fish oil appeared to improve subjective appetite and dampen the inflammatory response. The effectiveness of the intervention is influenced by gender and adherence. Males appear to be more responsive to the primary outcome variables than females, and the quality of response is improved with better adherence. These results provide evidence to support future interventions aimed at reducing the effects of PEW in dialysis patients.

Resumo: Em peixes, o 'beta'- glucano apresenta uma potente função imunoestimulante sendo cada vez maior a sua utilização como suplemento alimentar, aumentando significantemente a resistência à exposição infecciosa. Este prebiótico tem função de prevenir a colonização de patógenos por intensificar a ativação de macrófagos, proporcionando benefícios ao trato gastrointestinal e resultando em melhor desempenho e resistência a doenças. Este estudo teve a finalidade de avaliar os efeitos do 'beta' - glucano adicionado às rações peletizadas e extrusadas sobre o desempenho produtivo, indicadores de estresse, perfil hematológico e sobrevivência do pacu. Este experimento foi conduzido no Laboratório de peixes ornamentais, do Centro de Aqüicultura da UNESP (CAUNESP), em Jaboticabal, São Paulo. Foram utilizados 640 juvenis de pacu, com 24,7 ± 2,0 g, distribuídos em 32 aquários de vidro (130L). Os parâmetros físico - químicos da água foram mensurados quinzenalmente. Os peixes foram alimentados duas vezes ao dia, uma pela manhão e outra ao fim do dia. Os experimentos apresentaram um delineamento experimental inteiramente casualizado em esquema fatorial 2 x 4, utilizando níveis de inclusão do 'beta'- glucano de: 0 (controle), 0,1%, 0,2% e 0,3% por kg/ ração. Os níveis de'beta'-glucano avaliados neste estudo, não proporcionaram ganhos significativos no desempenho produtivo de juvenis de pacu, porém o tratamento 0,3% apresentou melhores resultados no GP, PF e TCE. A administração do 'beta'- glucano na dieta, durante todo período experimental, provocou alterações nos parâmetros hematológicos e indicadores de estresse do pacu. Os peixes alimentados com o 'beta'-glucano apresentaram maior resistência à infecção da bactéria A. hidrophila. Sendo assim, o tratamento 0,1% apresenta um custo/kg inferior e garante eficácia na saúde de juvenis de pacu.
Abstract: In fish, glucano has shown a potent immunostimulant function. The use of glucano is increasing significantly the resistance to diseases after infectious exposition. This prebiotic may be prevent the bacterial colonization, and activated macrophages, been beneficial to the digestive tract, resulting in better performance and disease resistance. This study will evaluate the glucano effects added in palletized and extruded diets of fish, analyzing fish perfOrmance, stress indicators, hematological profile and survival of pacu. This study was driven in Laboratory of ornamental fish, on Centro de Aqüicultura of UNESP (CAUNESP), in Jaboticabal, São Paulo. Were used 640 pacu juveniles, with 24,7 ± 2,0 g, distributed in 32 aquarium (130 L). The physical and chemical water parameters were measured every two weeks. Fish were fed twice a day, in the morning and another at the end of the day. In this trial were used 640 pacu juveniles (24.7 ± 2.0 g) distribuided in 32 aquariums (20 fish/aquarium). Throughout the experimental period, water remained at 26.5 oC and the others limnological parameters (dissolved oxygen, pH, alkalinity, ammonia and conductivity) stayed within normal values for the specie. The experimental trial design was entirely casualized in factorial scheme 2 x 4, evaluating two proceeding of diets (extruded and pelletized) and four 'beta' - glucan levels in diets: 0 (control), 0.1%, 0.2% and 0.3% with four repetitions. In this study,'beta' - glucan levels do not provide significant gains on pacu juveniles performance, but treatment with 0,3% - glucan showed better results of weight gain, weight final and specific growth rate. The administration of glucan in the diet, caused changes in hematological parameters and stress indicators in pacu. The fishes fed with glucan showed greater resistance to infection with A. hidrophila. Thus, treatment with 0,1% of glucan presented a lower cost/kg and shows efficiency in health of pacu juveniles.
Orientador: João Batista Kochenborger Fernandes
Coorientador: Elisabeth Criscuolo Urbinati
Banca: Sérgio Fonseca Zaiden
Banca: Fabiana Pilarski
Mestre

Organisms are continuously exposed to heterogeneous micro- and macro-parasitic species, hence simultaneous infections often occur in wild and farm environments. This joint project aimed to develop a co-infection model between chronic and acute infections, evaluating their impact on the fish immune system. Proliferative Kidney Disease was studied on farmed rainbow and brown trout during natural seasonal outbreaks, using a parasite gene (Tetracapsuloides bryosalmonae RPL18) as a proxy for assessment of parasite burden. In hosts with elevated susceptibility PKD pathogenesis was shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activity. Pathogen-free brown trout were exposed to Viral Haemorrhagic Septicaemia Virus (comparatively using European VHSV-Ia and North American VHSV-IVb strains) or to the bacterium Yersinia ruckeri. This European native species was highly resistant to the VHSV-IVb strain, which was undetectable in internal organs despite raising a strong antiviral and mucosal immune response. Following VHS and Yersiniosis infection, haemo-lymphopoietic organs were screened by RT-qPCR to assess the specific pathogen burdens and characterise the immune responses elicited. Transcription patterns were analysed for Interferons, CXC chemokines, SOCS (potential disease resistance biomarkers) and genes of the PACAP system. Lastly, PKD-infected brown trout were co-infected with VHSV-Ia, resulting in typical lesions while showing reduced and delayed mortality. PKD+/VHS+ fish were identified by RT-qPCR and histopathology screening. Pro-inflammatory and antimicrobial peptide genes were modulated following virus co-infection when compared to fish with single infection, with an earlier activation of cellular and humoral responses, and a stronger up-regulation of TH1 and antiviral genes. Oligonucleotide microarrays were used to assess the broader immune gene transcription modulation between single- and co-infected fish. Overall, the results suggest that the immune response of brown trout might be enhanced during the PKD/VHS co-infection.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Em peixes, o 'beta'- glucano apresenta uma potente função imunoestimulante sendo cada vez maior a sua utilização como suplemento alimentar, aumentando significantemente a resistência à exposição infecciosa. Este prebiótico tem função de prevenir a colonização de patógenos por intensificar a ativação de macrófagos, proporcionando benefícios ao trato gastrointestinal e resultando em melhor desempenho e resistência a doenças. Este estudo teve a finalidade de avaliar os efeitos do 'beta' - glucano adicionado às rações peletizadas e extrusadas sobre o desempenho produtivo, indicadores de estresse, perfil hematológico e sobrevivência do pacu. Este experimento foi conduzido no Laboratório de peixes ornamentais, do Centro de Aqüicultura da UNESP (CAUNESP), em Jaboticabal, São Paulo. Foram utilizados 640 juvenis de pacu, com 24,7 ± 2,0 g, distribuídos em 32 aquários de vidro (130L). Os parâmetros físico – químicos da água foram mensurados quinzenalmente. Os peixes foram alimentados duas vezes ao dia, uma pela manhão e outra ao fim do dia. Os experimentos apresentaram um delineamento experimental inteiramente casualizado em esquema fatorial 2 x 4, utilizando níveis de inclusão do 'beta'– glucano de: 0 (controle), 0,1%, 0,2% e 0,3% por kg/ ração. Os níveis de'beta'-glucano avaliados neste estudo, não proporcionaram ganhos significativos no desempenho produtivo de juvenis de pacu, porém o tratamento 0,3% apresentou melhores resultados no GP, PF e TCE. A administração do 'beta'- glucano na dieta, durante todo período experimental, provocou alterações nos parâmetros hematológicos e indicadores de estresse do pacu. Os peixes alimentados com o 'beta'-glucano apresentaram maior resistência à infecção da bactéria A. hidrophila. Sendo assim, o tratamento 0,1% apresenta um custo/kg inferior e garante eficácia na saúde de juvenis de pacu.
In fish, glucano has shown a potent immunostimulant function. The use of glucano is increasing significantly the resistance to diseases after infectious exposition. This prebiotic may be prevent the bacterial colonization, and activated macrophages, been beneficial to the digestive tract, resulting in better performance and disease resistance. This study will evaluate the glucano effects added in palletized and extruded diets of fish, analyzing fish perfOrmance, stress indicators, hematological profile and survival of pacu. This study was driven in Laboratory of ornamental fish, on Centro de Aqüicultura of UNESP (CAUNESP), in Jaboticabal, São Paulo. Were used 640 pacu juveniles, with 24,7 ± 2,0 g, distributed in 32 aquarium (130 L). The physical and chemical water parameters were measured every two weeks. Fish were fed twice a day, in the morning and another at the end of the day. In this trial were used 640 pacu juveniles (24.7 ± 2.0 g) distribuided in 32 aquariums (20 fish/aquarium). Throughout the experimental period, water remained at 26.5 oC and the others limnological parameters (dissolved oxygen, pH, alkalinity, ammonia and conductivity) stayed within normal values for the specie. The experimental trial design was entirely casualized in factorial scheme 2 x 4, evaluating two proceeding of diets (extruded and pelletized) and four 'beta' - glucan levels in diets: 0 (control), 0.1%, 0.2% and 0.3% with four repetitions. In this study,'beta' – glucan levels do not provide significant gains on pacu juveniles performance, but treatment with 0,3% – glucan showed better results of weight gain, weight final and specific growth rate. The administration of glucan in the diet, caused changes in hematological parameters and stress indicators in pacu. The fishes fed with glucan showed greater resistance to infection with A. hidrophila. Thus, treatment with 0,1% of glucan presented a lower cost/kg and shows efficiency in health of pacu juveniles.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The indiscriminate use of antimicrobial drugs in aquaculture may cause development of drug-resistant bacteria, which become more difficult to be controlled and eliminated. Probiotics may represent an alternative prophylactic disease control, replacing the use of antibiotics. In this respect, this study isolated, tested and identified potential probiotic bacteria from the gut of cobia, Rachycentron canadum, a potential candidate for marine aquaculture. 40 animals were captured, 10 from a private hatchery and 30 from an offshore culture system (PE, Brazil) between November 2010 and July 2011. Fishes from the hatchery had weight of 139.30 ± 31.52 g and length of 27.13 ± 1.46 cm, while those from offshore culture system weighed 456.77 ± 264.46 g and had length of 37.29 ± 6.05 cm. 45 bacterial were isolated and tested in vitro against five known pathogenic species, Aeromonas hydrophila (IOC/FDA 110-36), Pseudomonas aeruginosa (ATCC 15442), Streptococcus agalactiae (ATCC 13813), Vibrio parahaemolyticus (ATCC 17802) and Vibrio vulnificus (ATCC 27562). Fifteen strains (33.33%) had antibacterial activity to at least one pathogen, while eight (17.77%) were inhibited all pathogens tested. There was significant difference (P<0.05) between percentage of potential probiotic obtained from different seasons, rainy and dry. Strains that presented the best results antagonism test in vitro were identified as Lactobacillus plantarum, Bacillus coagulans, Klebsiella spp., Bacillus circulans, Lactococcus lactis subsp. lactis and Bacillus firmus. The largest inhibition zone observed in the antagonism test was produced by B. circulans against V. vulnificus. Some potential probiotic species identified were characterized by others authors, but isolated from the intestine of other fish species. It is suggested in vivo antagonism tests are performed to prove the effectiveness of these bacteria as probiotic to cobia.
O uso indiscriminado de drogas antimicrobianas na aquicultura pode contribuir para o desenvolvimento de bactérias resistentes aos medicamentos, o que torna esses micro-organismos mais difíceis de serem controlados e eliminados. Probióticos podem representar uma alternativa profilática no controle de doenças, em substituição ao uso de antibióticos. Neste contexto, o estudo foi realizado com o objetivo de isolar, testar e identificar bactérias com potencial probiótico do beijupirá, Rachycentron canadum, potencial candidato para a piscicultura marinha. Foram coletados 40 animais, 10 na fase de berçário e 30 na fase de engorda em sistema offshore (PE, Brasil), no período de novembro de 2010 a julho de 2011. Os peixes coletados do berçário apresentaram peso de 139,30 ± 31,52 g e comprimento de 27,13 ± 1,46 cm, enquanto os animais provenientes do sistema offshore apresentaram peso de 456,77 ± 264,46 g e comprimento de 37,29 ± 6,05 cm. Foram obtidos 45 isolados bacterianos testados in vitro frente a cinco espécies patogênicas conhecidas, Aeromonas hydrophila (IOC/FDA 110-36), Pseudomonas aeruginosa (ATCC 15442); Streptococcus agalactiae (ATCC 13813); Vibrio parahaemolyticus (ATCC 17802) e Vibrio vulnificus (ATCC 27562). Quinze isolados (33,33%) apresentaram atividade antibacteriana a pelo menos um patógeno e oito (17,77%) inibiram todos os patógenos testados. Houve diferença significativa (P<0,05) na proporção de isolados potenciais probióticos obtidos nas distintas épocas do ano, chuvosa e seca. Os isolados que apresentaram os melhores resultados no teste de antagonismo in vitro foram identificados como Lactobacillus plantarum, Bacillus coagulans, Klebsiella spp., Bacillus circulans, Lactococcus lactis subsp. lactis e Bacillus firmus. O maior halo de inibição observado no teste de antagonismo foi produzido por B. circulans frente ao V. vulnificus. Algumas espécies potenciais probióticas identificadas já foram caracterizadas por outros autores, porém isoladas de intestino de outras espécies de peixes. Sugere-se a realização de testes de antagonismo in vivo para que seja comprovada a efetividade das bactérias como probióticas para o beijupirá.

碩士
國立交通大學
分子醫學與生物工程研究所
107
Chronic Kidney Disease (CKD) affects 11.9% of adult population in Taiwan. CKD not only affects people's health but also costs large amount of money to provide CKD patient medical resources from the national government. Other complications are problems that can happen with CKD patient such as diabetes, hypertension, and cardiovascular diseases, etc. These complications can lead to a rapid deterioration of the disease and the depletion of existing medical resources. Therefore, early diagnosis and prevention of CKD become more important. In recent years, studies have pointed out that the microbial flora of the intestine is closely related to many diseases. The microbial flora of the intestine is not only co-evolved with humans, but also can affect the health of humans, synthesis of the body's metabolites, inhibition of intestinal pathogens, and reduce the toxicity of toxic substances on the human body has established a good immune system. But not all bacteria are beneficial to humans. Some anaerobic bacteria break down the metabolites of protein synthesis in the intestine but are toxic to humans. Previous studies have confirmed that derivatives of bacterial metabolites, "indoxyl sulfate" are highly accumulated in the blood of patients with chronic kidney disease, and are toxic to vascular cells, accelerating the deterioration of renal function. Therefore, it is more important to evaluate the amount of Indole. By searching for the bacteria that produce Indoxyl-sulfate to design a detection kit, develop a method to evaluate the ability of bacteria to produce Indole to detect the condition of end-stage chronic kidney patients, hoping to be in CKD patients found representative bacteria. It is expected that by changing the intestinal flora and significantly slow down the development of chronic kidney disease and effectively improve the health status of patients.

Juvenile chinook salmon (Oncorhynchus tshawytscha) were experimentally infected with Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease (BKD), to investigate the effects of BKD on three aspects of juvenile salmonid performance: (1) predator avoidance ability; (2) smoltification; and (3) physiological responses to stress. For these experiments, fish with different Rs-infection profiles (created by using an immersion challenge method) were sampled to assess physiological change and subjected to various performance tests during disease progression. When equal numbers of Rs-challenged and unchallenged fish were subjected to predation by northern pikeminnow (Ptychocheilus oregonensis) or smallmouth bass (Micropterus dolomieui), Rs-challenged fish were eaten in significantly greater numbers than controls by nearly two to one. A progressively worsening infection with Rs did not alter the normal changes in gill ATPase and condition factor associated with smoltification in juvenile chinook salmon. A dramatic proliferation of BKD was associated with maximal responses of indicators of smoltification, suggesting that the process of smoltification itself can trigger outbreaks of disease. When Rs-infected fish were subjected to three 60-s bouts of severe handling that were separated by 48-72 h, this experience did not lead to higher infection levels or increased mortality when compared to diseased fish that did not receive the stressors. Furthermore, the kinetics of plasma cortisol, glucose, and lactate over 24-h following each stressor were similar between fish with moderate to high BKD and those that had low or no detectable infection. Fish with moderate to high Rs infections had higher titers of cortisol and lactate prior to each application of the stressor and were also unable to consistently elicit a significant hyperglycemia in response to the stressors when compared to fish with low infection levels. During all experiments, fish consistently developed decreased hematocrits and blood glucose levels and increased levels of cortisol and lactate as the disease worsened, indicating that BKD is stressful, particularly during the later stages. Collectively, these results illustrate the impact of BKD on juvenile salmonids and have also ascribed some ecological significance to this disease beyond that of direct pathogen-related mortality.
Graduation date: 1999

Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD), is tolerant of recommended ultraviolet (UV) doses used in recirculation aquaculture systems (RAS). Membrane filtration (MF) is used to remove pathogens from wastewater in many industries, but has not been thoroughly tested in RAS. In this study, bacterial removal efficiencies were assessed between MF and UV treatments over 30 days in an RAS. Bacterial removal efficiencies were not significantly different (p > 0.05) between MF and UV treatments, which removed 98.5 ± 0.4 % and 99.6 ± 0.1 % of total bacteria and 93.2 ± 5.2 % and 92.5 ± 4.1 % of heterotrophic bacteria, respectively. Under laboratory conditions, a MF system was challenged with concentrated doses of F. psychrophilum and achieved 5.8 ± 0.2 log reductions of the pathogen. Therefore, MF represents a potential alternative to UV irradiation and could be used to reduce the prevalence of F. psychrophilum in RAS, potentially reducing the incidence of BCWD and its impact on the aquaculture industry.
Environment Canada and Ontario Ministry of Agriculture, Food and Rural Affairs.