RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

Population structure and genetic diversity of hatchery stocks as revealed by combined mtDNA fragment sequences in Indian major carp, Catla catla.

Catla catla is the second most important Indian major carp due to high growth rate and acceptance to consumers for food value. It is widely cultured in the Indian subcontinent as monoculture or polyculture. In the present study, genetic diversity among hatchery stocks (total 218 samples of catla) collected from different geographical regions of India was examined using mtDNA fragment sequence of Cyt b (306 bp) and D loop (710 bp). High numbers (57) of population specific haplotypes were observed in the present study. The results revealed significant genetic heterogeneity for the sequence data (FST = 0.27546, p < .05). Analysis of molecular variance revealed significant genetic differentiation among different catla populations. The information generated in present study could be useful to develop broad genetic base populations of catla.

Rapid recovery of complete mitogenome of Indian major carp, Catla catla from low depth paired end Illumina sequencing.

Here we report the reconstruction of the catla (Catla catla) complete mitochondrial genome sequence from low depth paired end Illumina sequencing. The genome is of 16,597 bp in size. Similar to other vertebrate mtgenomes, it consists of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and a putative control region. The present mtgenome is 3 bp longer than the earlier reported catla mtgenome from our laboratory. Majority of the mitochondrial genes are encoded by the H-strand. Phylogenetics analysis revealed that Catla catla is closer to Labeo rohita than other labeo species. Present study demonstrated the power of next generation sequencing towards hassle free and rapid sequencing of mitochondrial genomes of non-model organisms.

Low-depth shotgun sequencing resolves complete mitochondrial genome sequence of Labeo rohita.

Labeo rohita, popularly known as rohu, is a widely cultured species in whole Indian subcontinent. In the present study, we used in-silico approach to resolve complete mitochondrial genome of rohu. Low-depth shotgun sequencing using Roche 454 GS FLX (Branford, Connecticut, USA) followed by de novo assembly in CLC Genomics Workbench version 7.0.4 (Aarhus, Denmark) revealed the complete mitogenome of L. rohita to be 16 606 bp long (accession No. KR185963). It comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNAs and 1 putative control region. The gene order and organization are similar to most vertebrates. The mitogenome in the present investigation has 99% similarity with that of previously reported mitogenomes of rohu and this is also evident from the phylogenetic study using maximum-likelihood (ML) tree method. This study was done to determine the feasibility, accuracy and reliability of low-depth sequence data obtained from NGS platform as compared to the Sanger sequencing. Thus, NGS technology has proven to be competent and a rapid in-silico alternative to resolve the complete mitochondrial genome sequence, thereby reducing labors and time.

Preliminary genetic linkage map of Indian major carp, Labeo rohita (Hamilton 1822) based on microsatellite markers.

Linkage map with wide marker coverage is an essential resource for genetic improvement study for any species. Sex-averaged genetic linkage map of Labeo rohita, popularly known as 'rohu', widely cultured in the Indian subcontinent, was developed by placing 68 microsatellite markers generated by a simplified method. The parents and their F1 progeny (92 individuals) were used as segregating populations. The genetic linkage map spans a sex-averaged total length of 1462.2 cM, in 25 linkage groups. The genome length of rohu was estimated to be 3087.9 cM. This genetic linkage map may facilitate systematic searches of the genome to identify genes associated with commercially important characters and marker-assisted selection programmes of this species.

Assembly and variation analyses of Clarias batrachus mitogenome retrieved from WGS data and its phylogenetic relationship with other catfishes.

Whole genome sequencing (WGS) using next generation sequencing technologies paves the way to sequence the mitochondrial genomes with greater ease and lesser time. Here, we used the WGS data of Clarias batrachus, generated from Roche 454 and Ion Torrent sequencing platforms, to assemble the complete mitogenome using both de novo and reference based approaches. Both the methods yielded almost similar results and the best assembled mitogenome was of 16,510 bp size (GenBank Acc. No. KM259918). The mitogenome annotation resulted in 13 coding genes, 22 tRNA genes, 2 rRNA genes and one control region, and the gene order was found to be identical with other catfishes. Variation analyses between assembled and the reference (GenBank Acc. No. NC_023923) mitogenome revealed 51 variations. The phylogenetic analysis of coding DNA sequences and tRNA supports the monophyly of catfishes. Two SSRs were identified in C. batrachus mitogenome, out of which one was unique to this species. Based on the relative rate of gene evolution, protein coding mitochondrial genes were found to evolve at a much faster pace than the d-loop, which in turn are followed by the rRNAs; the tRNAs showed wide variability in the rate of sequence evolution, and on average evolve the slowest. Among the coding genes, ND2 evolves most rapidly. The variations present in the coding regions of the mitogenome and their comparative analyses with other catfish species may be useful in species conservation and management programs.

New encoded single-indicator sequences based on physico-chemical parameters for efficient exon identification.

The first step in gene identification problem based on genomic signal processing is to convert character strings into numerical sequences. These numerical sequences are then analysed spectrally or using digital filtering techniques for the period-3 peaks, which are present in exons (coding areas) and absent in introns (non-coding areas). In this paper, we have shown that single-indicator sequences can be generated by encoding schemes based on physico-chemical properties. Two new methods are proposed for generating single-indicator sequences based on hydration energy and dipole moments. The proposed methods produce high peak at exon locations and effectively suppress false exons (intron regions having greater peak than exon regions) resulting in high discriminating factor, sensitivity and specificity.

Derivation and characterization of embryonic stem-like cells of Indian major carp Catla catla.

Embryonic stem (ES)-like cells were derived from mid-blastula stage embryos of a freshwater fish, catla Catla catla, under feeder-free condition and designated as CCES cells. The conditioned media was optimized with 10% foetal bovine serum (FBS), fish embryo extract (FEE) having 100 µg ml(-1) protein concentration, 15 ng ml(-1) basic fibroblast growth factor (bFGF) and basic media containing Leibovitz-15, DMEM with 4·5 g l(-1) glucose and Ham's F12 (LDF) in 2:1:1 ratio using a primary culture of CCES cells. Cells attached to gelatin-coated plates after 24 h of seeding and ES-like colonies were obtained at day 5 onwards. A stable cell culture was obtained after passage 10 and further maintained up to passage 44. These cells were characterized by their typical morphology, high alkaline phosphatase activity, positive expression of cell-surface antigen SSEA-1, transcription factor Oct4, germ cell marker vasa and consistent karyotype up to extended periods. The undifferentiated state was confirmed by their ability to form embryoid bodies and their differentiation potential.

Derivation of rough attenuated variants from smooth virulent Aeromonas hydrophila and their immunogenicity in fish.

Aeromonas hydrophila is a Gram-negative bacteria associated with many disease conditions of aquatic animals. Two rough attenuated variants were derived from two smooth virulent types of this bacterium by continuous passage separately in laboratory over a period of 8 years in brain-heart infusion agar. These variants were checked to be rough lipopolysaccharides (LPS) types and attenuated since they neither produced any disease nor mortality in the injected fishes. The immunogenic potential of these variants remained unaltered as compared to parent smooth types and fish immunized with these variants resisted the infection and mortality upon cross challenge. Because of their intact immunogenic potential and loss of virulence, they may be used as candidates for fish immunization.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita.

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Non-specific immune parameters of brood Indian major carp Labeo rohita and their seasonal variations.

Different non-specific immune parameters and their seasonal changes in brood Indian major carp Labeo rohita reared in two major freshwater aquaculture regions of India viz. West Bengal and Orissa were investigated. It was undertaken for 2 consecutive years and included three main seasons of a year such as summer (March-May), rainy (July-September) and winter (November-January). Total serum protein, albumin and globulin levels were not significantly different throughout the year (p>0.01). Serum lysozyme and myeloperoxidase activities were lower (7.26+/-0.87mg/ml and, 0.54+/-0.11 OD, respectively) in winter as compared to any other season of the year. The bacterial agglutination titer was higher (p<0.01) in the rainy season (8.70+/-1.70) compared to summer and winter seasons (3.40+/-0.60 and 4.00+/-0.89, respectively). Haemagglutination and haemolytic activities did not vary (p>0.01) throughout the year. In blood smears, lymphocyte percentage was higher (75-80%) as compared to those of neutrophil (10-15%) and monocytes (5-10%) but eosinophilic granulocytes were present only in few cases. The differential leucocyte count did not vary significantly (p>0.05) in any season. This study indicated that certain non-specific immune parameters of this species can be modulated at certain times of the year.

Passive transfer of maternal antibodies and their existence in eggs, larvae and fry of Indian major carp, Labeo rohita (Ham.).

Lack of immune competence in the early stages of life leads to severe mortality in larval stages of different fish species including Indian major carp (IMC). Investigation through indirect enzyme linked immunosorbent assay (ELISA) and agglutination test revealed a significant increase in specific serum antibody response in the brood fish of Indian major carp, Labeo rohita (Ham.) following immunisation with a virulent Aeromonas hydrophila bacterin 1 month prior to breeding, which was transferred to larvae through the egg. No significant differences (P > 0.05) in mean antibody levels in larvae at the 1st and 2nd weeks post-hatch was recorded while a slight rise in antibody level was observed in 3-week-old fry, perhaps due to exposure to A. hydrophila present in the aquatic environment. Immunised brood fish serum, egg and larval extracts in non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent western blot analysis revealed an antibody molecule of approximate molecular weight 210 kDa. On challenge with virulent A. hydrophila, a significant reduction in mortality was recorded in immunised larvae and fry (58.0, 43.75 and 37.14% in the 1st, 2nd and 3rd week, respectively) relative to control fish (87.0, 79.0 and 76.4% in 1st, 2nd and 3rd week, respectively). The present study indicated the role of maternally derived antibody in protection of hatchlings of Indian major carp against specific pathogens.

The immunomodulatory effects of tuftsin on the non-specific immune system of Indian Major carp, Labeo rohita.

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p

High antigenic cross-reaction among the bacterial species responsible for diseases of cultured freshwater fishes and strategies to overcome it for specific serodiagnosis.

Antigenic sharing among the most commonly bacterial pathogens such as Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens of Indian major carps has been studied using immunological reactions such as cross-agglutination, disc diffusion and indirect enzyme linked immunosorbent assay (ELISA). The data were analysed using statistical analysis (SAS), version 6.12. The results showed high antigenic similarities among the bacterial whole cells, whole cell lysates, somatic 'O' antigens, lipopolysaccharides (LPS) and extracellular products (ECP). However, few or no similarities were observed in ECP components of <20kD. The present study indicates a need to develop differential diagnostic methods based on serology by choosing the highly specific less cross-reactive ECP antigen.

Bath immunisation of spawn, fry and fingerlings of Indian major carps using a particulate bacterial antigen.

Larval mortality in Indian major carps is one of the major problems encountered in the pond culture system. The present investigation was carried out to investigate the proper age, duration of exposure, and optimum bacterin concentration for vaccinating rohu (Labeo rohita) and catla (Catla catla) at their early stages with a formalin killed Edwardsiella tarda bacterin suspension. The development of immunological competence was recorded with spawn of rohu and catla of 3 weeks of age exposed to a bacterin at a concentration 10(9) cfu ml(-1) for 15 min, where it persisted up to 4 weeks post vaccination. They showed significant resistance against challenge with virulent E. tarda bacteria. Significant antibody titre could be recorded in advanced fries and fingerlings exposed to 10(9) cfu/ml(-1) bacterin concentration for 45 and 60 min, respectively.