Piscine francisellosis, caused by Francisella noatuenensis subsp orientalis (Fno), is an emerging infectious disease in the tilapia industry, but no effective commercial treatments or vaccines are available. The use of immunostimulants is a promising method to control diseases in aquaculture, and various algae and algal-derived compounds are potent immunostimulants for improving immune status. Algae produce a great variety of secondary metabolites that exert a broad spectrum of biological activities. The aim of this thesis was to evaluate the effectiveness of algal compounds against Fno in vitro and in vivo and determine their potential to control francisellosis infection in Nile tilapia Oreochromis niloticus L. under experimental conditions, and in an alternative host, namely the greater wax moth Galeria mellonella. Some of the algae and their compounds (Chlorella sp., alginic acid, and ß-glucan) exerted antimicrobial activity in vitro against Fno, Aeromonas hydrophila and Streptococcus agalactiae and stimulated responses of Nile tilapia macrophages (Chapter 2). An immersion challenge model for Fno STIR-GUS-F2f7 was developed in two genetic groups of Nile tilapia, and the homo gold strain was more susceptible to infection than wild type (Chapter 3). In vivo trials were conducted in Nile tilapia homo gold where fish were fed diets supplemented with 10% Scenedesmus quaricauda, 10% Haematococcus pluvialis, and 0.1% or 0.2% alginic acid or ß-glucan, and then challenged with Fno and co-infected with S. agalactiae (Chapter 4). The Fno challenge failed to produce mortality; however, co-infection resulted in high mortalities in all groups. As the in vivo trial in tilapia could not be to repeated, a G. mellonella model for Fno was validated. Fno doses between 0.7–1.7 x 108 CFU mL-1 killed G. mellonella, while tetracycline, alginic acid and ß-glucan rescued the wax moth from lethal doses of bacteria (Chapter 5).

Orientador: Germano Francisco Biondi / Resumo: A franciselose em peixes podem causar sérias perdas econômicas aos produtores. Os objetivos dessa pesquisa foram estimar e identificar a prevalência de Francisella noatunensis orientalis em Oreochromis niloticus cultivadas em tanques-rede na bacia hidrográfica do rio Araguari – Minas Gerais – Brasil. Foram coletadas aleatoriamente 150 amostras de baço de Oreochromis niloticus oriundas de cultivos em tanques-rede localizados em reservatórios de hidrelétricas localizadas na bacia hidrográfica do rio Araguari - Minas Gerais, Brasil e abatidas em três frigoríficos distintos. Foram mensurados o comprimento padrão (cm) e a massa corpórea (g) dos peixes. Antes da coleta do baço, foi identificado o sexo dos peixes. O baço foi utilizado para identificar a Francisella spp. pelo método de qPCR. As fêmeas apresentaram uma massa corpórea e um comprimento padrão menores significativamente (p<0,0001) quando comparados com os machos. Não foi observada relação significativa entre essas variáveis com a presença da bactéria (p>0,05). Os resultados da qPCR identificaram a Francisella spp. em seis amostras, correspondendo a prevalência de 4 %. Dessas, duas foram encaminhadas para a realização do sequenciamento do gene iglC pertencente à região 16S do RNA ribossômico, o qual identificou a presença de Francisella noatunensis orientalis em ambas. Esse achado de prevalência pode levar à instauração, por parte dos órgãos governamentais, de condutas de vigilância dos casos de franciselose no país, devi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Francisellosis in fishes can cause serious economic losses to producers. The aims of this research were to estimate and verify the prevalence of Francisella noatunensis orientalis in Oreochromis niloticus grown in cages system in Araguari river hydrographic basin – Minas Gerais state – Brazil. It was collected, randomly, 150 spleen samples of Oreochromis niloticus derived crops in cages located in hydroelectric reservoirs placed in Araguari river hydrographic basin – Minas Gerais state – Brazil, from three different slaughterhouses. It was measured the fishes standard lenght (cm) and the body mass (g). Before the collection of the spleen, the gender of the fishes was identified. The spleen was used to identify the Francisella spp. by qPCR method. The female showed a body mass and significantly smaller standard lenght (p<0,0001) when compared with males. It was not observed a significant relationship between these variables with the presence of bacterium (p>0,05). The qPCR results identified Francisella spp. in six samples, representing a prevalence of 4 %. From these, two were directed to the achievement of the sequencing of the gene iglC belonging to the 16S ribosomal RNA identified the presence of Francisella noatunensis orientalis on both. This prevalence finding may lead to the establishment, from government departments, monitoring behavior of francisellosis cases in Brazil, due to major economic losses generated by its etiological agent. / Doutor

Piscine francisellosis in an infectious emerging bacterial disease that affects several marine and fresh water fish species worldwide, including farmed salmon, wild and farmed cod, farmed tilapia and several ornamental species, for which no commercial treatment or vaccine exists. During 2011 and the first semester of 2012, chronic episodes of moderate to high levels of mortality with nonspecific clinical signs, and widespread multifocal white nodules as the most consistent gross pathological lesion were experienced by farmed tilapia fingerlings at two different locations in Northern Europe. In this study such outbreaks of granulomatous disease were diagnosed as francisellosis with a genus-specific PCR, and 10 new isolates of the bacterium including the one named STIR-GUS-F2f7, were recovered on a new selective “cysteine blood-tilapia” agar and cysteine heart agar with bovine haemoglobin. Ultrastructural observations of the pathogen in Nile tilapia (O. niloticus) tissues suggested the secretion of outer membrane vesicles (OMVs) by the bacterial cells during infection in these fish. This represented the first documented report of isolation of pathogenic Francisella strains from tilapia in Europe. The phenotypic characterisation indicated that isolates recovered were able to metabolise dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate and glucose-6-phosphate. The predominant structural fatty acids of the isolates were 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%) and 18:0 (5.5%). Anti-microbial resistance analyses indicated that STIR-GUS-F2f7 was susceptible to neomycin, novobiocin, amikacin, ciprofloxacin, imipenem, gatifloxacin, meropenem, tobramycin, nitrofurantoin, and levofloxacin using the quantitative broth micro-dilution method, while the qualitative disc diffusion method indicated susceptibility to enrofloxacin, kanamycin, gentamicin, tetracycline, oxytetracycline, florfenicol, oxolinic acid and streptomycin. The use of the following housekeeping genes: mdh, dnaA, mutS, 16SrRNA-ITS-23SrRNA, prfB putA rpoA, rpoB and tpiA indicated 100% similarity with other isolates belonging to the subspecies F. noatunensis orientalis (Fno). Koch’s postulates were successfully fulfilled by establishing an intraperitoneal injection (IP) challenge model with STIR-GUS-F2f7 in Nile tilapia. Moreover, the challenge model was used to investigate the susceptibility of 3 genetic groups of tilapia to STIR-GUS-F2f7. The lowest amount of bacteria required to cause mortality was 12 CFU/ml and this was seen as early as only 24 hours post infection in the red Nile tilapia and in the wild type after 26 days, no mortalities were seen in the species O. mossambicus with this dose. The mortality in red O. niloticus was significantly higher than that of the other two tilapia groups when 12 and 120 CFU/fish were injected. It was also observed that when a dose of 1200 CFU/ml was used, the mortality in O. niloticus wild type was significantly lower than that of the other two tilapia groups and no differences were seen among the 3 groups when the highest dose (1.2 x105 CFU/fish) was used. The median lethal dose (LD50) of O. niloticus wild type was the most stable during the experiment (values around 104 CFU/ml) and the highest of the three groups after day 25 post infection. At the end of the experiment (day 45) the LD50 was 30 CFU/ml in the red Nile tilapia, 2.3x104 CFU/ml for the wild type and 3.3x102 CFU/ml for O. mossambicus. This pattern, where the LD50 of the red tilapia was lower than that of the other two groups, was observed during the whole experiment. The outcomes of these experiments suggested that the red Nile tilapia family appeared to be the most susceptible while the wild type Nile tilapia family the most resistant. The complete genome of STIR-GUS-F2f7 was sequenced using next generation sequencing (NGS) Illumina Hi-Seq platform™, and the annotation of the assembled genome predicted 1970 protein coding sequences and 63 non-coding rRNA sequences distributed in 328 sub-systems. The taxonomy of the species Francisella noatunensis was revised using genomic-derived parameters form STIR-GUS-F2f7 and other strains in combination with a polyphasic approach that included ecologic, chemotaxonomic and phenotypic analyses. The results indicated that STIR-GUS-F2f7 and all the other strains from warm water fish represent a new bacterial species for which the name Francisella orientalis was assigned. Moreover the description of F. noatunensis was emended and the creation of a new subspecies within this taxon i.e. Francisella noatunensis subsp. chilense was proposed. The results of this study led to the development of a highly efficacious vaccine to protect tilapia against francisellosis.