42 March 2012 lation of survival to aeromoniasis with myeloperoxidase, superoxide and lysozyme activities; negative correlations with bacterial agglutination titer, haemolysin titer and haemagglutination titer; and a significant positive correlation with ceruloplasmin level among different rohu families. Because negatively correlated parameters cannot be applied as a selection criterion, only the acute-phase protein ceruloplasmin could, potentially, serve as a marker trait for resistance to aeromoniasis. The first generation resistant line of rohu showed a significantly higher level of response in the case of respiratory burst activity of blood phagocytes, serum myeloperoxidase activity and ceruloplasmin level compared to the susceptible line (Sahoo et al. 2011). However, the traits should be heritable to be incorporated into a selection program. Hence, in this scenario, the use of immunological markers in this selection program is of questionable value. Molecular Techniques Selection generally depends on genetic variation to disease resistance (Lund et al. 1995). Molecular markers, such as single nucleotide polymorphism (SNP) markers and microsatellites, are providing new tools for selection programs. Comparison of molecular markers identified from resistant and susceptible lines can give information about the polymorphic loci that govern resistance. Association of markers with any trait (QTL) can lead to identification of genes responsible for resistance or susceptibility. Selection based on marker data is called Marker Assisted Selection (MAS) and, if a gene is used, the process is called Gene Assisted Selection (GAS). Genotyping different families and correlation with survival will further confirm the identified polymorphic markers. Screening individuals can be achieved with a SNP chip. A SNP chip is only available for an Atlantic salmon breeding population as a potential genomic tool for selecting for resistance to amoebic gill disease (AGD) in a breeding program run by the Salmon Enterprises of Tasmania (Saltas) (Dominik et al. 2010). Selection of breeders can be based on the presence of individual markers or causative genes (QTL), rather than phenotypes of sibs only. Pedigree relationships could be replaced by genomic similarities estimated through information on genetic markers (Odegard et al. 2011). Genomic selection yields high genetic gain, accuracy of selection, and lower rates of inbreeding (Sonesson and Meuwissen 2009). Incorporation of identified QTLs in the selection program has already started in Scotland (Houston et al. 2008) and Norway (Moen et al. 2009) for the selection of Atlantic salmon against infectious pancreatic necrosis virus. Presently only one group in the Central Institute of Freshwater Aquaculture, India, in collaboration with NOFIMA, Norway is trying to identify molecular markers for disease resistance against aeromoniasis in rohu. The transcriptome sequence generated 330,327 SNPs, and indels and fixed allele differences between the resistant and susceptible line were detected. Ninety-six SNP loci contained allele frequency differences of 1.0, while 104 SNP loci contained allele frequency differences > 0.95 between the lines. MH class I antigen and galactoside-binding soluble lectin 9 gene showed fixed allele frequency differences between susceptible and resistant lines (Robinson et al., in manuscript). Constraints and Future Directions Initially selection was based on survival in ponds. Subsequently artificial challenge experiments in controlled and semi-controlled environments were used to select individuals or families based on resistance to a particular disease. Although few selection programs on disease resistance to various pathogens are active, each approach has drawbacks and practical difficulties. The following points of concern should be considered during an experimental challenge test, where large numbers of fish are subjected to challenge from a particular pathogen: 1. There are many strain variations within a particular bacterial pathogen such as A. hydrophila. Therefore, before conducting a challenge test, the isolate should be selected on the basis of thorough biochemical and molecular characterization of the pathogen. The organism should be highly virulent and have an array of toxic protein expression. 2. Inasmuch as the challenge test must be conducted over a period of years, it is essential for estimation of heritability to maintain pathogen virulence for a long period for subsequent challenge testing. 3. The appropriate route of challenge and the consistency in obtaining lethal dose are crucial. 4. A direct challenge study needs skilled manpower, a separate challenge facility, and is time consuming. 5. For a family-based selection program, it is often difficult to get similar-size fish from all families at the same time, which may influence the challenge picture. 6. Environmental factors, including temperature and climatic conditions, may influence bacterial pathogenicity. Fig. 3. Focal necrosis of liver in A. hydrophila-infected rohu (H & E × 100). (Continued on page 44)
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