Diversity Analysis of the Immunoglobulin M Heavy Chain Gene in Nile Tilapia, Oreochromis niloticus (Linnaeus)

A full-length cDNA encoding the immunoglobulin (IgM) heavy chain gene of Nile tilapia was successfully cloned using the 5’ and 3’ RACE techniques. The complete cDNA of the Nile tilapia IgM heavy chain gene is 1,921 bp in length and has an open reading frame (ORF) of 1,740 bp, which corresponds to 580 amino acid residues. The deduced amino acid sequence of the Nile tilapia IgM heavy chain includes a typical secretory IgM heavy chain designated "On-sIgM" and a variable region that is connected to 4 constant regions to form the LH-VH-Cµ1-Cµ2-Cµ3-Cµ4 pattern. Comparisons of the nucleotide and amino acid sequences of On-sIgM with IgM heavy chains of other organisms showed the highest similarity scores of 62.6 and 55.4%, respectively, to the orange-spotted grouper (Epinephelus coioides). Structural analysis of 126 cDNAs encoding variable domains of the IgM heavy chain revealed that at least 9 VH families, 6 DH segments and 4 JH families were utilized using several mechanisms to generate the repertoire of antigen-binding domains. Variation analysis of the variable domains indicated that the amino acid sequences of the framework regions (FRs) were less variable than those of the complementarity determining regions (CDRs), among which the most variable was CDR3. Tissue expression profile analysis using quantitative real-time RT-PCR of healthy Nile tilapia showed that the IgM heavy chain gene was ubiquitously expressed in all 13 tested tissues, but the highest expression level was observed in the head kidney, followed by the spleen, intestine and peripheral blood leukocytes (PBLs). Furthermore, Southern blot analysis of the constant region of the IgM heavy chain gene of 3 different fishes indicated that Nile tilapia genomes may contain 2 copies of the IgM gene.Keywords: Nile Tilapia, IgM Heavy Chain, Variable Region, Diversity, Secreted Form, Southern Blo

Competition on Using Nutrient for Growth between Bacillus spp. and Vibrio harveyi

ABSTRACT Competition, by using nutrient for growth, between Bacillus pumilus NW01, B. sphaericus NW02 and B. subtilis NW03 and Vibrio harveyi in vitro was studied by culturing each bacteria in Nutrient Broth (+ 1.5% NaCl). The initial concentration of 10 2 CFU/ml in monoculture and co-culture of Bacillus spp. and V. harveyi was used. Total Bacillus and Vibrio counts were conducted after 0, 24, 48, 72, 96 and 120 hours. B. pumilus NW01, B. sphaericus NW02 and B. subtilis NW03 decreased V. harveyi by 39.10, 43.62 and 34.46%, respectively. Antagonistic properties of Bacillus spp. against V. harveyi in vivo was tested by feeding shrimp with spores (10 11 -10 12 CFU/g) of each Bacillus and their mixture at 5 g/kg for 1 month. The amount of Vibrio spp. in the intestine of all Bacillus treated shrimp decreased by 20.97-32.45 % as compared with the control. The results showed that these Bacillus spp. could be applied as an effective probiotic in Penaeus monodon culture

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Acknowledgements We are highly grateful to thank Seppic, France, for providing us with the commercial products of MontanideTM ISA 763A VG and MontanideTM 437 ISA 763B VG. Funding This research project was financially supported by Mahasarakham University (Grant No. 6517022Peer reviewedPostprin

Identification and Antibiotic Sensitivity Test of the Bacteria Isolated from Tra Catfish (Pangasianodon hypophthalmus [Sauvage, 1878]) Cultured in Pond in Vietnam

ABSTRACT Tra catfish (Pangasianodon hypophthalmus [Sauvage, 1878]) cultured in Vietnam is one of the most important products for export to international market such as U.S.A., Japan and Europe. The bacterial isolation was conducted from pond-cultured Tra catfish in the provinces with intensive culture system. There were a total of 97 isolates from 65 diseased fish. Sixty-six isolates from 97 isolates were identified by biochemical tests. They were consisted of Edwardsiella ictaruli (58 isolates [87.9%]), Aeromonas hydrophila (2 isolates [3.03%]) and 6 unidentified isolates (9.07%). Antibiotic sensitivity test was also conducted to determine the drugs that could be used to control these two bacteria. Effective drugs that showed good sensitivity against E. ictaluri were ciprofloxacin, amoxycillin and ampicillin, florfenicol, doxycycline and oxytetracycline (in order of number of isolates that showed sensitive). Two isolates of Aeromonas hydrophila were sensitive to sulphamethoxazole, ciprofloxacin, oxytetracycline, enrofloxacin, erythromycin, sulphamethoxazole/trimethoprim, doxycycline and florfenicol

Effect of putative probiont Enterococcus hirae on the haematological parameters of juvenile African catfish, Clarias gariepinus (Burchell, 1822) during pre- and post-challenge against Aeromonas hydrophila

Probiotics have been widely known to have the ability to improve the immune system of livestock and aquatic animal. The present study was carried out to evaluate the effect of dietary supplementation of two probiotic isolates of Enterococcus faecium on hematological parameters of juvenile African catfish, Clarias gariepinus during pre- and post-challenge with aquatic pathogen, Aeromonas hydrophila. The probiotics were previously isolated from vegetable wastes (mung bean sprouts, Vigna radiate and cucumber, Cucumis sativus) which have been fermented for 7 days. The experimental fish (270 tails) with an average weight of 5.13 ± 1.03 g were distributed and divided randomly into i) control (30 tails), fed with commercial diet ii) E1 (30 tails), fed with diets supplemented with 108 CFU/ml of E. faecium isolated from fermented cucumber, iii) E2 (30 tails), fed with 108 CFU/ml of E. faecium isolated from fermented mung bean sprouts. The feeding trial was conducted for 50 days. All experimental groups were then challenged with A. hydrophila (1.5 × 106 CFU/mL) via intraperitoneal injection on day 51. Prior to challenge, blood samples were collected from five fish randomly selected from each group on the day 51 (pre-challenge). After 72 hours of post-challenge, blood samples were again collected from five fish from each groups. The hematological parameters such as total erythrocyte count (RBC), total leucocyte count (WBC), packed cell volume (PCV), hemoglobin (Hb), the derived blood indices of mean corpuscular volume (MCV) and corpuscular hemoglobin concentration (MCHC) were examined. Hematological profiles of pre- and post-challenge infected juvenile catfish were compared with the control groups. The RBC, Hb, WBC, PCV, MCV and MCHC of fish fed with probiotics showed higher significant difference (P<0.05) as compared to control groups during pre- and post-challenge of pathogen. The high level of RBC and WBC during pre- and post-challenge showed the capability of the probiotics to improve the immune response of juvenile African catfish and thus increased the fish disease resistance against A. hydrophila infection. The result suggested that E. faecium could be used effectively as a probiotic in aquaculture

Characterization of the Major Histocompatibility Complex Class II Genes in Miiuy Croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3–85.7% and 11.0–88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes

Characterization, Stress Response and Functional Analyses of Giant River Prawn (Macrobrachium rosenbergii) Glucose-Regulated Protein 78 (Mr-grp78) under Temperature Stress and during Aeromonas hydrophila Infection

The endoplasmic reticulum (ER) is an organelle important for several functions of cellular physiology. This study identified the giant river prawn’s glucose-regulated protein 78 (Mr-grp78), which is important for ER stress mechanisms. Nucleotide and amino acid analyses of Mr-grp78, as compared with other species, revealed the highest similarity scores with the grp78 genes of crustaceans. An expression analysis by quantitative RT-PCR indicated that Mr-grp78 was expressed in all tissues and presented its highest expression in the ovary (57.64 ± 2.39-fold), followed by the gills (42.25 ± 1.12), hindgut (37.15 ± 2.47), thoracic ganglia (28.55 ± 2.45) and hemocytes (28.45 ± 2.26). Expression analysis of Mr-grp78 mRNA levels under Aeromonas hydrophila induction and heat/cold-shock exposure was conducted in the gills, hepatopancreas and hemocytes. The expression levels of Mr-grp78 in these tissues were highly upregulated 12 h after bacterial infection. In contrast, under heat- and cold-shock conditions, the expression of Mr-grp78 was significantly suppressed in the gills at 24–96 h and in the hepatopancreas at 12 h (p &lt; 0.05). A functional analysis via Mr-grp78 gene knockdown showed that Mr-grp78 transcription in the gills, hepatopancreas and muscle strongly decreased from 6 to 96 h. Furthermore, the silencing of this gene effectively increased the sensitivity of the tested prawns to heat- and pathogenic-bacterium-induced stress. The results of this study clearly demonstrate the significant functional roles of Mr-grp78 in response to both temperature and pathogen treatments

Molecular Identification and Dual Functions of Two Different CXC Chemokines in Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1&ndash;10 &mu;g strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro