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

Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein–protein docking modelling suggests the helveticin-J homolog may be able to block VP28–PmRab7 interactions and interrupt WSSV infection

Genotypic Study of Pneumocystis jirovecii in Human Immunodeficiency Virus-Positive Patients in Thailand

Pneumocystis jirovecii is one of the common opportunistic infections in human immunodeficiency virus (HIV)-infected patients in Thailand. Information regarding genotypic and epidemiological of this organism in Thai patients is not available. We analyzed the genotypes of 28 P. jirovecii-positive specimens from bronchoalveolar lavage and sputum samples from HIV-infected Thai patients based on nucleotide variations of the internal transcribed spacer regions 1 and 2 of the rRNA gene. Thirteen genotypes were the same as previously reported outside Thailand. Ten genotypes, which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb, and Qq, were new. Ir and Rp were unique and dominant types observed in HIV-infected Thai patients. Thirteen specimens (46.4%) were infected with a single type of P. jirovecii, and fifteen (53.6%) were mixed infections. These differences may be used as genotypic markers for studying the epidemiology and transmission of P. jirovecii in the Thai population

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

Clonal diversity of the glutamate dehydrogenase gene in <it>Giardia duodenalis </it>from Thai Isolates: evidence of genetic exchange or Mixed Infections?

Abstract Background The glutamate dehydrogenase gene (gdh) is one of the most popular and useful genetic markers for the genotypic analysis of Giardia duodenalis (syn. G. lamblia, G. intestinalis), the protozoan that widely causes enteric disease in humans. To determine the distribution of genotypes of G. duodenalis in Thai populations and to investigate the extent of sequence variation at this locus, 42 fecal samples were collected from 3 regions of Thailand i.e., Central, Northern, and Eastern regions. All specimens were analyzed using PCR-based genotyping and recombinant subcloning methods. Results The results showed that the prevalence of assemblages A and B among these populations was approximately equal, 20 (47.6%) and 22 (52.4%), respectively. Sequence analysis revealed that the nucleotide diversity of assemblage B was significantly greater than that in assemblage A. Among all assemblage B positive specimens, the allelic sequence divergence within isolates was detected. Nine isolates showed mixed alleles, ranged from three to nine distinct alleles per isolate. Statistical analysis demonstrated the occurrence of genetic recombination within subassemblages BIII and BIV was likely. Conclusion This study supports increasing evidence that G. duodenalis has the potential for genetic exchange.</p