Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

The primary vector of the trypanosome parasite causing human and animal African trypanosomiasis in Uganda is the riverine tsetse fly Glossina fuscipes fuscipes (Gff). Our study improved the Gff genome assembly with whole genome 10× Chromium sequencing of a lab reared pupae, identified autosomal versus sex-chromosomal regions of the genome with ddRAD-seq data from 627 field caught Gff, and identified SNPs associated with trypanosome infection with genome-wide association (GWA) analysis in a subset of 351 flies. Results from 10× Chromium sequencing greatly improved Gff genome assembly metrics and assigned a full third of the genome to the sex chromosome. Results from ddRAD-seq suggested possible sex-chromosome aneuploidy in Gff and identified a single autosomal SNP to be highly associated with trypanosome infection. The top associated SNP was ~1200 bp upstream of the gene lecithin cholesterol acyltransferase (LCAT), an important component of the molecular pathway that initiates trypanosome lysis and protection in mammals. Results suggest that there may be naturally occurring genetic variation in Gff in genomic regions in linkage disequilibrium with LCAT that can protect against trypanosome infection, thereby paving the way for targeted research into novel vector control strategies that can promote parasite resistance in natural populations.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system. These genes protect bacteria from being eaten by bacteriophages. In this study, CRISPR-Cas systems were characterized using a genomic approach. For this purpose, genome sequences of Lactobacillus johnsonii strains were retrieved, and the diversity, occurrence, and evolution of the CRISPR-Cas systems were analyzed. Then, homology analyses of spacer sequences in identified CRISPR arrays were performed to analyze and characterize the diversity of target phages and plasmids. Finally, the evolutionary paths of spaceromes in each subtype of CRISPR arrays were performed using acquisition and deletion events surveyed under the selective pressure of foreign plasmids and phages. Results showed that 138 strains contain valid CRISPR-Cas structures (CRISPR loci together with the Cas genes) in their genomes, which accounted for about 17% of the L. johnsonii studied strains belonging to subtypes II-A, I-E, and I-C. Moreover, results indicated that some specific groups of plasmids were targeted with specific CRISPR array systems. Homology analysis of spacer sequences with phage genomes also revealed that spacers of strains that harbored only CRISPR-Cas subtype-II targeted a greater diversity of foreign phages. In conclusion, the current study indicates that there is great diversity between the CRISPR-Cas systems identified in L. johnsonii strains. Such diverse CRISPR-Cas systems indicate that these systems are naturally active and important in terms of adaptive immunity and evolutionary relationships.