Making the most of its short reads: a bioinformatics workflow for analysing the short-read-only data of Leishmania orientalis (formerly named Leishmania siamensis) isolate PCM2 in Thailand

Background: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. Results: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. Conclusions: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

Background: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. Methods: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. Results: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. Conclusions: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite

Evaluation of <i>Bacillus</i> spp. as Potent Probiotics with Reduction in AHPND-Related Mortality and Facilitating Growth Performance of Pacific White Shrimp (<i>Litopenaeus vannamei</i>) Farms

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease affecting shrimp aquaculture worldwide. In this study, natural microbes were used in disease prevention and control. Probiotics derived from Bacillus spp. were isolated from the stomachs of AHPND-surviving Pacific white shrimp Litopenaeus vannamei (22 isolates) and mangrove forest soil near the shrimp farms (10 isolates). Bacillus spp. were genetically identified and characterized based on the availability of antimicrobial peptide (AMP)-related genes. The phenotypic characterization of all Bacillus spp. was determined based on their capability to inhibit AHPND-causing strains of Vibrio parahaemolyticus (VPAHPND). The results showed that Bacillus spp. without AMP-related genes were incapable of inhibiting VPAHPND in vitro, while other Bacillus spp. harboring at least two AMP-related genes exhibited diverse inhibition activities. Interestingly, K3 [B. subtilis (srfAA+ and bacA+)], isolated from shrimp, exerted remarkable inhibition against VPAHPND (80% survival) in Pacific white shrimp and maintained a reduction in shrimp mortality within different ranges of salinity (75–95% survival). Moreover, with different strains of VPAHPND, B. subtilis (K3) showed outstanding protection, and the survival rate of shrimp remained stable among the tested groups (80–95% survival). Thus, B. subtilis (K3) was further used to determine its efficiency in shrimp farms in different locations of Vietnam. Lower disease occurrences (2 ponds out of 30 ponds) and greater production efficiency were noticeable in the B. subtilis (K3)-treated farms. Taking the results of this study together, the heat-shock isolation and genotypic-phenotypic characterization of Bacillus spp. enable the selection of probiotics that control AHPND in Pacific white shrimp. Consequently, greater disease prevention and growth performance were affirmed to be beneficial in the use of these probiotics in shrimp cultivation, which will sustain shrimp aquaculture and be environmentally friendly

Trial Evaluation of Protection and Immunogenicity of Piscine Bivalent Streptococcal Vaccine: From the Lab to the Farms

Streptococcosis is one of the major diseases that causes devastation to farmed fish, leading to significant economic losses all around the world. Currently, two serotypes of Streptococcus agalactiae, serotype Ia and III, have been identified as virulent strains and major causative agents of the disease in farmed Nile tilapia (Oreochromis niloticus Linn.) in Thailand. Upon inactivated vaccine development, monovalent inactivated whole-cell vaccines demonstrated high specific antibody production against homologous serotypes and limited production with heterologous serotypes. However, for higher efficacy, a bivalent streptococcal vaccine was designed to maximize protective immunity to both serotypes. Interestingly, our bivalent vaccine could successfully induce specific antibody production against both serotypes with similar levels, and the response could extend over the 8 weeks of the experimental period. Evaluation of vaccines in the laboratory scale revealed relative percent survival (RPS) of vaccinated tilapia to serotype Ia (81.2 ± 9.4%) and serotype III (72.2 ± 4.8%), respectively. The efficacy of the bivalent vaccine showed significant RPS higher than the monovalent vaccine (p < 0.05) at 30 days, and the protection of all those vaccines was reduced thereafter. Evaluation of the vaccine in a farm trial in different locations in Thailand revealed the efficacy of the bivalent vaccine in increasing the production yield by greater than 80% in all tested farms in 2015 and 2021. Taken together, this study affirms the efficacy of the bivalent streptococcal vaccine in the prevention of streptococcus disease in Nile tilapia, which could be used in different areas. This vaccine development could be effectively applied in the tilapia culture industry

Microarray analyses of shrimp immune responses

Abstract Shrimp aquaculture is one of the major foodproducing industries in the world. However, it is being impacted by several problems including diseases, antibiotic use, and environmental factors. The extent of the effects of these problems in the immune system of the shrimp at the molecular level is just beginning to be understood. Here, we review the gene expression profile of shrimp in response to some of these problems using the high-throughput microarray analysis, including white spot syndrome virus, yellow head virus, Vibrio spp., peptidoglycan, oxytetracycline, oxolinic acid, salinity, and temperature

Anti-Protease Activity Deficient Secretory Leukocyte Protease Inhibitor (SLPI) Exerts Cardioprotective Effect against Myocardial Ischaemia/Reperfusion

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity

Comparative draft genomes of Leishmania orientalis isolate PCM2 (formerly named Leishmania siamensis ) and Leishmania martiniquensis isolate PCM3 from the southern province of Thailand

(1) Background: Autochthonous leishmaniasis, a sandfly-borne disease caused by the protozoan parasites Leishmania orientalis (formerly named Leishmania siamensis) and Leishmania martiniquensis, has been reported for immunocompromised and immunocompetent patients in the southern province of Thailand. Apart from the recent genomes of the northern isolates, limited information is known on the emergence and genetics of these parasites. (2) Methods: This study sequenced and compared the genomes of L. orientalis isolate PCM2 and L. martiniquensis isolate PCM3 with those of the northern isolates and other 14 Leishmania species using short-read whole-genome sequencing methods and comparative bioinformatic analyses. (3) Results: The genomes of the southern isolates of L. orientalis and L. martiniquensis were 30.01 Mbp and 32.39 Mbp, and the comparison with the genomes of the northern isolates revealed species-level similarity with a level of genome and proteome variation, suggesting the different strains. Comparative proteome analysis showed six protein groups with 53 unique proteins for the strain PCM2 and 97 for the strain PCM3. Certain proteins were related to virulence, drug resistance, and stress response. (4) Conclusion: Therefore, the findings could indicate the need for more genetic and population genomic investigation, and the close monitoring of L. orientalis and L. martiniquensis in Thailand and neighboring regions

Design of a Chimeric Multi-Epitope Vaccine (CMEV) against Both Leishmania martiniquensis and Leishmania orientalis Parasites Using Immunoinformatic Approaches

Leishmaniasis is a parasitic disease caused by protozoan flagellates of the genus Leishmania. Recently, Leishmania martiniquensis and Leishmania orientalis, emerging species of Leishmania, were isolated from patients in Thailand. Development of the vaccine is demanded; however, genetic differences between the two species make it difficult to design a vaccine that is effective for both species. In this study, we applied immuno-informatic approaches to design a chimeric multi-epitope vaccine (CMEV) against both L. martiniquensis and L. orientalis. We identified seven helper T lymphocyte (HTL) epitopes, sixteen cytotoxic T lymphocyte (CTL) epitopes, and eleven B-cell epitopes from sixteen conserved antigenic proteins found in both species. All these epitopes were joined together, and to further enhance immunogenicity, protein and peptides adjuvant were also added at the N-terminal of the molecule by using specific linkers. The candidate CMEV was subsequently analyzed from the perspectives of the antigenicity, allergenicity, and physiochemical properties. The interaction of the designed multi-epitope vaccine and immune receptor (TLR4) of the host were evaluated based on molecular dockings of the predicted 3D structures. Finally, in silico cloning was performed to construct the expression vaccine vector. Docking analysis showed that the vaccine/TLR4 complex took a stable form. Based on the predicted immunogenicity, physicochemical, and structural properties in silico, the vaccine candidate was expected to be appropriately expressed in bacterial expression systems and show the potential to induce a host immune response. This study proposes the experimental validation of the efficacy of the candidate vaccine construct against the two Leishmania

A virulent isolate of yellow head nidovirus contains a deformed envelope glycoprotein gp116

AbstractYellow head virus (YHV) is a highly virulent pathogen of penaeid shrimp. An isolate obtained from Penaeus vannamei during a yellow head disease outbreak in February 2006 in Ratchaburi Province, Thailand was purified following passage in experimentally infected shrimp. SDS-PAGE of purified virions indicated that envelope glycoprotein gp116 in the Ratchaburi/2006 isolate was smaller and relatively less abundant than in the Chachoengsao/1998 YHV reference strain. The variant gp116 reacted poorly in immunoblots with a gp116 mouse monoclonal antibody and a rabbit anti-serum to a baculovirus-expressed, C-terminally truncated, [His]6-tagged gp116 that reacted strongly with gp116 of the homologous Chachoengsao/1998 strain. The anti-gp116 polyclonal serum also failed to neutralise the infectivity of the Ratchaburi/2006 isolate in in-vivo assays conducted in P. vannamei, but effectively neutralised the infectivity of the reference strain. Sequence analysis of the ∼6.0 kb structural protein gene region and 3′UTR of the Ratchaburi/2006 isolate indicated >99.9% overall nucleotide identity with the Chachoengsao/1998 strain. However, in Ratchaburi/2006 a deletion in ORF3, corresponding to 54 amino acids near the N-terminal signal peptidase cleavage site of gp116, resulted in the loss of six conserved cysteine residues and two predicted N-glycosylation sites. Analysis of this ORF3 region in 25 viruses representing each of the six genotypes in the yellow head complex identified this modified form of gp116 in two other virulent YHV isolates classified as genotype 1b. The data indicate that, although the deletion causes a significant structural deformation of gp116 which reduces its incorporation into virions and eliminates the major neutralisation sites, the virus remains highly infectious, virulent and fit for survival