Aims
This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and ...virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems.
Methods and Results
Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas.
Conclusions
Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand.
Significance and Impact of the Study
We identified two major serotypes of S. agalactiae isolates associated with the outbreak in tilapia culture in Thailand. We developed multiplex PCR assays for 14 virulence genes, which may be used to predict the pathogenicity of the isolates and track future infections. Multiplex PCR typing of the GBS virulence genes was developed and might be further used to predict the pathogenicity of S. agalactiae.
This study elucidates the immune modulation including the expression of cytokine genes following dietary administration of three selected probiotic bacteria—
Lactobacillus rhamnosus,
Enterococcus ...faecium and
Bacillus subtilis to fish, rainbow trout
Oncorhynchus mykiss. They were fed for 45 days on either a basal control diet or one of the three probiotic diets containing the specific bacteria in freeze-dried form at a density of 10
9
CFU
g
feed
−1.
The non-specific immune parameters examined—superoxide anion production by the head kidney leukocytes and the alternate complement activity of serum was improved by probiotic feeding. Besides this, the relative gene expressions of interleukin-1
β1, tumor necrosis factor 1 and 2 and transforming growth factor-
β were up regulated in the spleen and the head kidney. The comparatively better performance of
E. faecium could possibly be linked to their suitable ambient temperature conditions. Thus, probiotic bacteria delivered in feed exerts its influence on the immune system of fish, both at cellular and molecular levels.
Aims
To investigate the virulence of the Vp_PirAB‐like genes in Vibrio parahaemolyticus‐ acute hepatopancreatic necrosis disease (AHPND)‐causing strain and the factors that are associated with the ...virulence level.
Methods and Results
The virulence of Vp_PirAB‐like was examined using a non‐virulent strain FP11 of V. parahaemolyticus transformed with a plasmid harbouring Vp_PirAB‐like genes and then it was used to challenge shrimp Litopenaeus vannamei and Marsupenaeus japonicus. Both species experienced 100% mortality at 10 days post infection. Analysis of a mutant strain (E1M), that was originally identified as virulent strain (E1) but lost its virulence to L. vannamei, revealed that it lacked a part of the Vp_PirA‐like gene and all of the Vp_PirB‐like gene. The copy numbers of Vp_PirA‐like and Vp_PirB‐like genes varied among virulent strains and were not correlated with their virulence. In Western blotting, Vp_PirA‐like and Vp_PirB‐like proteins were detected in both the cell lysate and the culture supernatant. The strongest intensity of detecting band in the culture supernatant was observed in the strain that caused the highest mortality. The V. parahaemolyticus AHPND‐causing strain, unlike the human tdh‐positive strain, did not show any enterotoxicity.
Conclusion
Vibrio parahaemolyticus AHPND‐causing strains secrete the Vp_PirA‐like and Vp_PirB‐like proteins during the growing phase. The amount of secreted proteins affects the shrimp mortality.
Significance and Impact of the Study
The secreted proteins of Vp_PirAB‐like are key factors of virulence in the V. parahaemolyticus AHPND‐causing strain, but not gene copy.
Aims
Genomic characterization of Harveyi clade vibrio strain Y6 causing ‘Scale drop and Muscle Necrosis syndrome’ (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam.
Methods and Results
A ...bacterial genome was sequenced using Illumina MiSeq platform. Multilocus sequence analysis confirmed that the bacterium belongs to Vibrio harveyi species. Further phylogenetic analysis inferred from core genome SNPs revealed a close relationship between our bacterium and the V. harveyi isolated from groupers in Taiwan and China. blastp results indicated that V. harveyi piscine strains carried numerous adhesin, secretion system, siderophore and toxin‐related genes. Genome comparison between Y6 and 32 strains of V. harveyi from different origins showed that at least 17 potential virulence genes were present exclusively in the strain Y6. Many of these (six of 17 genes) were homologous to pyoverdine siderophore, a secreted high‐affinity iron chelator, clusters originally found in Pseudomonas aeruginosa. Genome of V. harveyi Y6 was incorporated by a bacteriophage VHY6φ and replication protein of the phage was most similar to CTXφ described previously in Vibrio cholerae and Vibrio fischeri. However, the cholera toxin‐encoding genes, namely ctxA and ctxB, were absent from VHY6φ, while the CTXφ‐enterotoxin gene (zonula occludens toxin; zot) remained intact.
Conclusions
Several putative virulence genes and a phage carrying toxin gene were identified in the genomes of SDMN‐associated V. harveyi Y6.
Significance and Impact of the Study
This study confers genomic information of the piscine pathogenic V. harveyi which recently caused widespread mortality. Such information is of importance to gain insight into bacterial molecular pathogenesis.
Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon. A total of 1062 expressed sequence tag (EST) clones were sequenced ...unidirectionally. ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered. They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively. Several types of each AMP homologue were found. Sequence alignments of ALF in P. monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini. Only one major type of penaeidin (penPm3) was found in P. monodon. In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized. Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes. Expression of these antimicrobial peptides in P. monodon subjected to V. harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins.
Neobenedenia melleni is a monogenean parasite that causes significant mortality and economic losses in fish aquaculture. Changes in the antigenic composition of this parasite occur during its ...developmental stages. In this study, we evaluated humoral parameters in serum and transcriptional immune responses of yellowtail naturally infected with N. melleni. In addition, in vitro assays were performed to study the stimulatory effects of antigens from larvae and adults on spleen leucocytes from non‐infected fish at 6 and 24 h post‐stimulation. The results showed enhanced total protein, myeloperoxidase and antiprotease activities in N. melleni‐infected fish compared with non‐infected ones. The induction of Toll‐like receptors (TLRs) and pro‐inflammatory cytokines in spleen leucocytes during natural infection with N. melleni suggests that these immune‐related genes play an important role in the initiation of the immune defence mechanism for controlling parasite infection. Interestingly, the magnitude of in vitro responses of spleen leucocytes was dependent on the parasitic stage. An important stimulation of gene expression by adult antigens on spleen leucocytes was observed. Differential expression patterns of TLRs and target cytokines in yellowtail leucocytes in both in vivo and in vitro studies suggest that the quality of yellowtail immune response is conditioned by N. melleni development.
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