Étude du polymorphisme du gène majeur d’histocompatibilité de classe IIb (MHIIb) chez l’omble de fontaine (Salvelinus fontinalis)

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes.

Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974-1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Phi(ST) = -0.00296-0.00284), increasing considerably at the height of stocking (1980s Phi(ST) = 0.18932) and decreasing post-stocking (1990s/2002 Phi(ST) = 0.04934-0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.

Estudo da doença de inverno em dourada (Sparus aurata L.) de cultivo a sul de Portugal

Dissertação de mest. em Aquacultura, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2003

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease XbaI, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.

Avaliação de sistema de cultivo integrado, a partir da reciclagem de águas residuais submetidas a tratamento primário: pesquisa de espécies dos gêneros Salmonella, Shigella, Vibrio e Aeromonas

Para avaliar um sistema integrado de aquicultura foram realizadas análises microbiológicas da água utilizada neste sistema e determinada a incidência e resistência antimicrobiana dos enteropatógenos no ecossistema relacionado. As amostras de água testadas apresentaram 32,9% de taxas de coliformes fecais (≤1.600/100mL), de acordo com a OMS para piscicultura em águas residuais. Salmonella spp. foram detectadas em 14,5% das amostras. De um total de 33 cepas, 15,1% eram resistentes a um ou dois antimicrobianos testados e resistência a múltiplas drogas não foi observada. Aeromonas spp. foram identificadas em 91,6% das amostras. De um total de 416 cepas, resistência a uma classe de antimicrobianos foi observada em 66,3% e a multirresistência às drogas em 37,7%. Na avaliação da virulência dos isolados de Aeromonas hydrophila, 85,3% das cepas apresentaram Beta-hemólise nos três diferentes tipos de eritrócitos empregados e 99,1% nos eritrócitos de coelho e cavalo, sendo possível a caracterização através da PCR do gene aerA e lip, em 100% das amostras. Os resultados obtidos apontam para a relevância quanto às vantagens da implementação de um sistema integrado, disponibilizando alimentos com custo reduzido, porém este sistema necessita de um controle rígido e efetivo para que estes produtos não constituam veículos para a disseminação de doenças.

Distribuição e conservação dos genes que codificam as proteínas VgrG e Hcp em espécies de Aeromonas

Aeromonas spp. são bastonetes Gram negativos amplamente distribuídos nos ambientes aquáticos, com relatos de isolamento em água de abastecimento público e alimentos. Este micro-organismo possui potencial de causar doenças intestinais e extraintestinais cuja patogenicidade está associada a sua virulência multifatorial. Diversos determinantes de virulência de Aeromonas já foram identificados, incluindo sistemas de secreção de proteínas. O sistema de secreção tipo VI (SST6) é o mais recente sistema de secreção de proteínas identificado em bactérias cuja presença em estirpes no gênero Aeromonas pode implicar atividades de citotoxicidade para o hospedeiro, pois esse sistema é capaz de injetar moléculas efetoras dentro da célula, interferindo diretamente nos processos celulares. A fim de determinar a presença e analisar a distribuição dos genes hcp e vgrG codificadores das proteínas efetoras do SST6 em Aeromonas spp. o presente estudo examinou 119 cepas isoladas de diversas origens pela técnica da PCR após o desenho de oligonucleotídeos iniciadores específicos. Objetivamos ainda analisar a variabilidade genética interespecífica dos genes hcp e vgrG a partir de dados de sequenciamento. Os resultados obtidos indicaram a distribuição dos genes vgrG e hcp em 46% das cepas de Aeromonas hydrophila e Aeromonas caviae de diferentes origens. Entre as cepas de A. hydrophila a maior frequência foi observada nas cepas isoladas de humanos, onde todas foram positivas para os iniciadores que amplificaram um produto de 541 pb do gene vgrG e 418 pb do gene hcp. Entre as cepas de A. caviae, a incidência de genes vgrG e hcp foi mais elevada nas cepas isoladas de alface (60%) e peixes (50%). As cepas analisadas de origem ambiental apresentaram índice total de 36% de positividade, apresentando frequência de 60% e 22% em A. hydrophila e A. caviae, respectivamente. Os dados obtidos da análise de cepas de origem alimentar mostraram a presença dos genes vgrG e hcp em 67% (A. hydrophila) e 60% (A. caviae) das cepas isoladas de folhas de alface. Nas cepas isoladas de queijo os genes foram encontrados em 67% e 12,5% das cepas de A. hydrophila e de A. caviae, respectivamente. O alinhamento múltiplo entre as sequências dos segmentos dos genes hcp e vgrG obtidas no sequenciamento indicou grau de identidade nucleotídica de 75 a 100% entre as sequências de hcp e 80 a 100% entre as sequências de vgrG. Em conclusão, nossos resultados indicaram que os iniciadores desenhados foram capazes de detectar suas sequências alvo em cepas de A. caviae e outras espécies de Aeromonas, sugerindo a existência de homologia entre os genes nas diferentes espécies, confirmada após sequenciamento de DNA. Os dados indicaram que esses genes estão distribuídos em várias espécies de Aeromonas e em cepas isoladas de diversas fontes. Ressaltamos a prevalência de cepas de A. hydrophila PCR-positivas em isolados clínicos, sugerindo a participação do SST6 no complexo universo da virulência multifatorial que permeia esse micro-organismo

Capacidade de invasão, persistência intracelular e atividade citotóxica de cepas de Aeromonas spp. em modelo celular in vitro e ex vivo

As Aeromonas são consideradas patógenos em potenciais para o homem e animais e estão amplamente distribuídas no ambiente sendo a água e os alimentos importantes veículos de transmissão. Muitos estudos têm demonstrado que a patologia causada pela infecção por Aeromonas é complexa e envolvem inúmeros fatores de virulência, dentre eles a aderência, invasão, enterotoxinas, hemolisinas, exoenzimas, sideróforos, flagelos, formação de biofilme e mecanismos de secreção. No presente estudo, analisamos os mecanismos de patogênese mediados por A. caviae e A. hydrophila, avaliando a participação desses microrganismos nos processos de adesão, invasão, persistência intracelular e citotoxidade celular. Foram utilizados ensaios quantitativos in vitro para testar associação, invasão e persistência intracelular em linhagens celulares HEp-2 e/ou T84. A interação de tecidos intestinais de coelho cultivados in vitro (IVOC) com três cepas de A. caviae originárias de fezes diarréicas também foi avaliada. Observamos que 10 (62,5%) das 16 cepas de Aeromonas spp. de diferentes origens, submetidas aos testes de invasão quantitativos foram capazes de invadir células HEp-2 e T84 em 6 horas de incubação. As cepas positivas nos testes de invasão foram submetidas ao teste quantitativo de persistência em células HEp-2 e sobreviveram no ambiente intracelular por 48 e/ou 72 horas sem multiplicação. A interação de três cepas de A. caviae com a mucosa intestinal de coelho ex vivo resultou em aderência, produção de muco e alterações como, intensa vacuolização e drástica desorganização estrutural que levaram a destruição das microvilosidades intestinais. Este estudo demonstrou que subconjuntos de cepas de A. caviae e A. hydrophila de diversas origens, foram capazes de invadir, persistir ou destruir linhagens celulares in vitro. Nosso estudo também evidenciou que cepas de A. caviae causaram expressivas alterações morfológicas que resultaram na destruição de epitélios intestinais de coelho ex vivo. Finalmente, nossos resultados contribuíram para reforçar o potencial patogênico de cepas de Aeromonas, em especial, as de origem vegetal e clínica.

Survival of antibiotic resistant Aeromonas strains in different water conditions

Studies were conducted to find out the survival of three antibiotic resistant Aeromonas strains in different types of water. The selected Aeromonas strains were A. hydrophila (local), A. sobria (local) and A. hydrophila (Thai), which were only recovered from farmed fishes. Seven types of water were used. Among these experimental water, lake water, distilled water and fish farm pond water had supported the long time survival of A. hydrophila. In contrast, private fish farm pond water was the most favorable for A. sobria. Deionized water was found not to support the survival of any species but Masjid pond water and FRI pond water were found to be moderately suitable for all the species. However, the survival pattern of Thai strain of A. hydrophila was found to have similarity with the survival of the local strain of A. hydrophila.

Pathogenicity of Aeromonas hydrophila artificially infected to Indian major carp (Cirrhinus mrigala Ham.)

Abrasion, feeding, injection and immersion methods were used to evaluate the pathogenicity of five different strains of Aeromonas hydrophila viz. RG (rui gill), ML (mrigal lesion), SG (sharpunti gill), F1K (mrigal kidney), GFL (gold fish lesion) and Ah-19 (Aeromonas hydrophila-19, Ref. Strain) against C. mrigala H. Bacterial suspension containing viable cells of 7.5x 10⁵ per ml was found to be very effective in intramuscular injection and feeding resulting 100% mortality after 96hr of inoculation. The strain RG, ML and F1K produced scale loss with erosion of the skin surface with/without hemorrhagic lesion after 48hr of inoculation following abrasion method. The strains SG and Ah-19 resulting scale loss with erosion of the skin surface with/without hemorrhagic lesion after 72hr of inoculation following abrasion and injection methods. SG and F1K caused reddening in mouth region after 72hr of feeding inoculation, whereas RG resulted frank ulcers from eroded dermal layer exposing underlying musculature which was hemorrhagic after 96hr of inoculation by abrasion method.

Pathogenicity of Aeromonas sobria to Thai silver barb (Barbodes gonionotus Bleeker) and its sensitivity to some antibiotic agents

Five isolates of Aeromonas sobria, collected from the diseased fish were selected for detection the pathogenicity following water-born infection method on silver barbs (Barbodes gonionotus) at the selected exposure dose 2.5x10⁸ CFU/ml which was standardized by preliminary test. In the experimental condition lesion and mortality were found in fishes. Among the isolate, Ass17 Ass19, Ass31 and Ass36 were successfully infected 20-60% fishes. Another isolate Ass20 was found non-pathogenic. Drug sensitivity test was performed by six antibiotics viz. Oxytetracycline, Oxolinic acid, Chloramphenicol, Stilphamethozazole, Streptomycin, Erythromycin. All the isolates showed variable reaction patterns to antibiotics. Most of the isolates were found sensitive to Oxytetracycline (OT), Oxolinic acid (OA) and Chloramphenicol (C) but resistance to Erythromycin and Sulphamethoxazole (SXT). Isolate Ass31 found resistant to Oxolinic acid.

In vitro phagocytic study of blood leucocytes and peritoneal macrophages of walking catfish Clarias batrachus ( Ham.) against Aeromonas hydrophila and Escherichia coli.

In observation of in vitro phagocytic activity against Aeromonas hydrophila isolate 34k (a virulent form) and Escherichia coli (an avirulent bacteria) of neutrophil- and monocyte-like cells of walking catfish Clarias batrachus showed phagocytosis. N eutrophils and monocytes phagocytized the avirulent form of bacterial isolate more than the virulent one. Other blood leucocytes did not show phagocytosis. Peritoneal macrophage of the fish were separated by glycogen elicitation and the macrophages were being adhered on plastic cover slips for studying their in vitro phagocytic activity. Most of the cells were alive after adherence and showed phagocytosis against the virulent and avirulent bacteria. The percent phagocytosis and phagocytic index were higher against the avirulent E. coli than the virulent A. hydrophila.

Experimental pathogenesis of Aeromonas hydrophila bacteria in shing Heteropneustes fossilis(Bloch)

Pathogenicity of Aeromonas hydrophila bacteria was tested on the stinging catfish Heteropneustes fossilis. Before artificial infection the morphological, biochemical and physiological characters of Aeromonas hydrophila were studied. The infections were done by two different methods, viz., intramuscular (IM) and intraperitoneal (IP) injection. In infection experiment, each group of 10 fish were injected either intramuscularly or intra peritoneally with one dose higher than the LD50 dose (9.6 x 107 CFU/fish). All the fish tested died within 1 to 9 days. Both in cases of intramuscular and intraperitoneal injection, external pathology were found. Haemorrhagic lesions were evident at the site of injection. The posterior end of the body surface was found to develop greyish-white lesion that was extended up to caudal fin. Hyperemic anal region and the fin bases were also observed. Total bacterial loads in liver, kidney and intestine were determined. Aeromonas hydrophila could be isolated from liver, kidney and intestine of the experimentally infected fish. In case of intramuscular injection the highest and the lowest bacterial load was found to be 2.4 x 107 CFU/g of liver and 2.1 x 102 CFU/g of kidney and in case of intraperitoneal injection they were found to be 3.6 x 106 CFU/g of kidney and 1.2 x 104 CFU/g of kidney respectively. It was concluded that A. hydrophila could cause serious disease condition to Heteropneustes fossilis and its pathogenesis in the fish was also very efficient.

Humoral and protective response of Indian major carps to immersion vaccination with Aeromonas hydrophila

Fry of the Indian major carps, Catta catla (Ham.), Labeo rohita (Ham.) and Cirrhinus mrigala (Ham.) were immunized at 4 and 8 weeks post hatching (wph) by direct immersion in a suspension (10 super(8) cells ml super(-1))of heat inactivated Aeromonas hydrophila. Following the same procedure, booster dose was administered 20 days after the first immersion. Antibodies as well as protective response produced in both the groups after the first and the booster immersion were different and significant (P<0.05). No significant difference was found between the species in the two age groups. The specimens immunized 8 wph showed higher antibody titres and protection than the 4 wph group. C. catla had higher relative percent survival followed by L. rohita and C. mrigala.