Soutenance mémoire
Les maladies bactériennes en aquaculture
RESISTANCE OF CHARR (Salvelinusfontinalis) AND (Salvelinus alpinus) TO OPPORTUNISTIC INFECTIONS IN SEA W ATER:
Fish protect themselves against pathogenic microorganisms by an immune system comparable to that of humans and other vertebrates (Anderson, 1990). The first line of defence is based on non specific immune mechanisms. If a pathogen penetrates into the organism, the resulting inflammation attracts phagocytic cells that destroy the invader. This natural resistance is normally effective enough to protect fish from infectious diseases until specific immune responses are being induced (Kollner et al. , 2002). If the non specific response is unsuccessful, the major histocompatibility complex (MllC) cannot activate the specific immune response by the presentation of antigenic peptides to T cells. To be recognized, a foreign antigen must be degraded into smalt antigenic peptides that form complexes with class 1 or class II MllC molecules. These molecules are structurally and functionally distinct cell surface glycoproteins involved in antigen presentation to T cells. Class 1 molecules, which are expressed on the surface of all nucleated cells are involved in the presentation of endogenously derived peptides to CD8-positive (cytotoxic) T lymphocytes. On the other hand, class II molecules, expressed mainly in the antigenpresenting cells (B lymphocytes, macrophages, and activated T cells) present exogenously derived peptides to CD4-positive (helper) T lymphocytes (Iwama and Nakanishi, 1996).
The MllC genes represent sorne of the most polymorphic genes, with multiple loci and a considerable number of alleles at each given locus (Grimholt et al. , 2002). They play an important role in disease resistance to pathogens by their efficacy to present a wide variety ofantigenic peptides derived from pathogens (Van Muiswinkel et al., 1999). It was recently found that the teleostean class 1 and class II genes reside on different linkage groups. Furthermore, several studies in salmonids have shown that each class contains one major and dominantly expressed locus (Grimholt et al., 2003).
Sequencing and identification of alleles
Our selected samples were sent to the sequencing services provided by the immunogenetic department at the Notre-Dame campus of the Centre hospitalier de l’Université de Montréal (CHUM) , which used an ABI 3100 DNA sequencer with 16 capillaries. Ali sequence alignments were carried out using ClustalW (European Bioinformatics Institute) and amino acids sequences were obtained using MEGA version 3.1 (Kumar et al., 2004). From the difTerent sequences found, our difTerent alleles were determined. Another PCR amplification from parental DNA was realized. Purified PCR products were sent to the sequencing services in order to determine genotypes and to confirm the difTerent alleles. By analysing the different chromatograms using Sequencher (Gene Codes Corporation), heterozygotes versus homozygotes individuals were determined.
Media preparation
Different selective culture medium were prepared and stocked at 4°C in order to facilitate the isolation and identification of the pathogens responsible of opportunists lesions affecting charr in salt water. Marine agar (MA) was used to detect total bacteria from water sampi es, TCBS for the detection of Vibrio spp, Anacker and Ordal agar (AOA) prepared with seawater (contained in grl: 5g tryptone, 0.5g yeast extract, O.2g beef extract, O.2g sodium acetate, 15g Agar, IL of salt water, pH: 7.2-7.4) for the detection of Flavobacterium spp (Anacker and Ordal, 1959) and Flexibacter maritimus Medium (FMM) also prepared with seawater (contained in grl : 5g peptone, 0.5g yeast extract, O.Olg sodium acetate, 15g agar, IL of salt water, pH : 7.2-7.4) for the detection of Flexibacter maritimus (Pazos et al., 1996).
Sampling
Two series of samplings were done: one in June at the beginning of the experiment and another in August at the end, to ensure that it was the same pathogen in both cases. Five sick fish were removed before feeding in the different tanks. These fish were transported alive to the laboratory and analysed directly upon arrivai to avoid degeneration of tissue that could cause an uncertain diagnostic. Fish were sacrificed by a blow on the head, adipose and caudal fins were sectioned and stored in 95% ethanol for future analysis whereas weight and forklength were measured. Externallesians were the mast prominent at gills, tale or tlank. These lesions were cleaned with 70% ethanol, then incised with a sterile scalpel. Sowing by scratches in aseptic conditions were made with an inoculation loop on the different culture medium. Triplicate plates labelled for each charr sampled were maintained in the dark at room temperature and frequently examined. Three isolations were realised to obtain a single type ofbacteria. We picked representative isolates of each colony type observed for characterization and identification. A drop of the suspended culture to be examined was transferred on a microscope slide with an inoculation loop and was stained using the 77730 Gram Staining Kit following the manufacturer’s indications (Fluka, NeuUlm, Germany). Representative isolates were removed, placed in aliquot of 1 ml containing 1501-11 of autoclaved water and stocked at -20°C. In order to confirm our preliminary results, a nested polymerase chain reaction system developed by Cepeda et al. (2003) was applied to detect Flexibacter maritimus from fish tissue. Clinical isolate of Flexibacter maritimus (ATCC 43398) provided by Uhland (Faculté de médecine vétérinaire, Université de Montréal, Canada) was used to test the sensitivity of the nested PCR proto col as reference strain. The stain was stored in vials at -80°C in Marine agar broth (Difco Laboratories, USA) with 20% glycerol added. DNA from the representative isolates was purified by Phenol: Chloroform: Isoamyl (Alcohol 25 : 24: 1 saturated with 10mM Tris, pH 8.0, ImM EDTA) (Sigma-Aldrich, Germany). The reference strain was used as positive control whereas an Escherichia coli DNA and a tube without any DNA constituted our negative controls.
Primers
According to the protocol described by Cepeda et al. (2003), two universal primers, 20F (5 ‘- AGAGTTTGATCATGGCTCAG-3 ‘) and 1500R (5 ‘-GGTTACCTTGTTACGACTT-3 ‘), complementary to conserved regions of most eubacterial 16S rRNA, were used for the first PCR step. For the second PCR, two specific primers MarI (5 ‘ TGTAGCTTGCTACAGATGA-3 ‘) and Mar2 (5 ‘-AAATACCTACTCGTAGGTACG-3’) expected to give a product of 400 bp were used. Nucleotides were synthesized by Biocorp Inc. (Montréal, Canada). The cycling conditions for PCRI were 30 cycles of denaturation (95°C for 30 seconds), annealing (57°C for 30 seconds), and extension (72°C for 60 seconds). A preheating step at 95°C for 5 minutes and a final extension step of 5 minutes at 72°C were done. The cycling conditions for PCR 2 consisted of a preheating step of 94°C for 2 min, followed by 40 cycles at 94°C for 1 second, 54°C for two seconds and 5 seconds at 72°C, followed by a final extension step of 72°C for 4 minutes. The PCR products were analysed on a 1 % agarose gel.
Based on an efficient specific medium selection, we were able to make an initial diagnosis of the pathogen affecting fish. FMM was chosen due to its ability to allow better growth of Tenacibaculum maritimum in comparison to the growth of heterotrophic halophilic bacteria, such as Vibrio, Pseudomonas, and Aeromanas species, which usually overgrow the plates (Avendaîi.o-Herrera et al., 2005). Results were then confirmed through the nested PCR protocol of Cepeda et al. (2003) considered as the most adequate for an accurate detection of Tenacibaculum maritimum in diagnostic pathology as weil as in epidemiological studies of gliding bacterial disease in marine fish (Avendaîi.o-Herrera et al., 2004).
The filamentous orgamsm Tenacibaculum maritimum (formerly, Cytophaga marina, Flexibacter marinus and Flexibacter maritimus) is the causative agent of marine flexibacteriosis, an important disease widely distributed in cultured marine fishes (Toranzo et al. , 2005). This etiological agent has the potential to cause severe mortalities in several fish species reared in seawater around the world (Avendano-Herrera et al. , 2005). It was first identified in Japan in 1979 (Bernadet, 1997) and was reported as a salt-water requiring organism producing infections characterized by eroded mouth and fins and skin lesions (Wakabayashi et al., 1986). Young fish suffer a more severe form of the disease than juveniles (Margarinos et al., 1995). The disease is influenced by a multiplicity of environmental (stress) and host-related factors (skin surface condition) and could be more severe if temperatures are above 15°C (Margarinos et al., 1995). Until recently, no vaccines were available to prevent the disease (Bernadet, 1997), except one for turbot. However, it is not effective in other marine fish (Santos et al., 1999).
In general, growth performance was important in ail families in seawater, and especially in S. alpinus families. Furthermore, in both groups intraspecific (AA, AR, RR) and interspecific (AA, CF, CC), condition factors were higher and differences reduced in September, which indicates that at the end of the experiment, ail families were in good condition. Our results showed that in September, resident S. fontinalis for intraspecific families and S. Alpinus – S. fontinalis for interspecific families were in better condition than others. The fact that anadromous S. fontinalis had lower condition factors than resident throughout the whole summer cou Id be explained by their physiology. Anadromous S. fontinalis are found to be more streamlined (narrower and shallower bodies) than resident brook trout (Morinville and Rasmussen, 2003), our data seemed to confirm it.
We isolated and characterized the parental class II ~ 1 alleles in brook and Arctic broodstocks. Alignment of the 21 alleles’ sequences revealed that most of the variability (76%) resides in the peptide-binding sites. Much of the variation found in the charr sequences corresponds to polymorphic sites of the ~ 1 domain in higher vertebrates. As Langefors et al. (2001) noted, this indicates that the polymorphic sites in the fish alleles differed only slightly from those in human alleles. The high variability of exon 2 was in accordance with observations made on mammalian class II~ genes (Ono et al., 1993). It is interesting to note that sorne alleles only differed by a single (d and f; n and m; h and i) or two nucleotides (d and e; i and j). The fact that most of them encoded for different amino acid sequences (at the exception of alleles h and i) confirmed the high variability of exon 2. In their study, Langefors et al., (2001) also found two different alleles that differed by a single nucleotide. They concluded that one of the se two alleles could be ‘an example of a new allele that has been arisen and been maintained through frequency-dependent selection due to its association with resistance to furonculosis’ (Langefors et al. , 2001).
Specific alleles to anadromous Arctic charr, anadromous and resident brook charr were found, as weil as alleles being shared between both species. Miller and Withler (1996) indicated that comparison of Mlle exons within and between closely related species often reveals the existence of shared allelic lineages, which indicates that the formation of alleles predated speciation .
The estimation of allelic combinations was realized from the parental genotype. The progeny inherits two genes, one from each parent, so the progeny proportion is Y4 for each allele. Nevertheless, future analysis for genotype identification of offspring will be planned and will confirm the allelic combinations. Based on the high level of heterozygosity from the parental generation, an important range of allelic combinations is expected to occur in the progeny. These data will allow to evaluate if progeny survival to infection was associated with specific alleles. In addition, paternal half-sib crosses will allow us to study the maternaI effect and to determine if genetic differences exist between resistant and susceptible individuals.
DISCUSSION GÉNÉRALE:
Le cadre général de cette étude dépasse un projet de maîtrise car au départ, il avait été conçu comme un projet de doctorat. La suite du projet sera par conséquent finalisée par d’ autres que moi. Dans le cadre de ma maîtrise, j’ai établi le répertoire des allèles du CMH classe np 1 chez les géniteurs, identifié l’ agent pathogène Flexibacter maritimus responsable d’infections opportunistes chez les ombles en milieu marin, noté des différences interfamiliales de résistance lors du transfert en eau de mer et suggéré des pistes d’ analyses pour la poursuite du projet.
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Table des matières
CHAPITRE 1. INTRODUCTION GÉNÉRALE
Le système immunitaire
Le complexe majeur d’histocompatibilité
Les maladies bactériennes en aquaculture
Le stress et la résistance aux maladies
Problématique et objectifs
CHAPITRE II. RESISTANCE OF CHARR (Salvelinus fontinalis) AND (Salvelinus alpinus) TO OPPORTUNISTIC INFECTIONS IN SEAWATER
Introduction
Material and methods
Fish
Parental genotypes ofMHC class II (region Pl) alleles
Identification of pathogens
Stress exposure
Statistical analysis
Results
Parental genotypes ofMHC class II (region Pl) alleles
Identification of pathogens
Stress exposure
Discussion
DISCUSSION GÉNÉRALE
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