RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.
CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.
METHODOLOGY/PRINCIPLE FINDINGS: Here we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection.
CONCLUSIONS/SIGNIFICANCE: In conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.