Contribution of introns to the species diversity associated with the apicomplexan parasite, Neospora caninum.

Neospora caninum is an intracellular parasite considered a leading cause of bovine reproduction failure worldwide, and a serious neurological disease of canines. Transplacental transmission in intermediate hosts is considered the most efficient means of transmission, which strictly involves asexual reproduction. Nonetheless, extensive genetic diversity has been reported within the species. What is yet to be elucidated are the major drivers of such diversity, and their impact on important parasite phenotypes such as virulence. Instead of protein-encoding sequences, genome and transcriptome data were used to investigate SNPs in introns between two distinct N. caninum isolates, with reported differences in pathogenicity. Variant analysis identified 840 and 501 SNPs within intergenic regions and introns, respectively, distinctly concentrated on chromosomes VI and XI, whereas the rest of the genome was monomorphic in comparison. Gene ontologies for SNP-dense intron-containing genes included ATP binding, transmembrane transport, protein kinase activity, and transcription and translation processes. This study shows that variation in non-coding DNA is contributing to N. caninum intraspecies genetic diversity, and potentially influencing and contributing to important parasite mechanisms. Finally, we present an assembled and annotated N. caninum apicoplast genome and show that this essential organelle is highly conserved between the two isolates, and related Coccidia.

Machine learning and applications in microbiology.

To understand the intricacies of microorganisms at the molecular level requires making sense of copious volumes of data such that it may now be humanly impossible to detect insightful data patterns without an artificial intelligence application called machine learning. Applying machine learning to address biological problems is expected to grow at an unprecedented rate, yet it is perceived by the uninitiated as a mysterious and daunting entity entrusted to the domain of mathematicians and computer scientists. The aim of this review is to identify key points required to start the journey of becoming an effective machine learning practitioner. These key points are further reinforced with an evaluation of how machine learning has been applied so far in a broad scope of real-life microbiology examples. This includes predicting drug targets or vaccine candidates, diagnosing microorganisms causing infectious diseases, classifying drug resistance against antimicrobial medicines, predicting disease outbreaks and exploring microbial interactions. Our hope is to inspire microbiologists and other related researchers to join the emerging machine learning revolution.

Species diversity and genome evolution of the pathogenic protozoan parasite, Neospora caninum.

Neospora caninum is a cyst-forming coccidian parasite of veterinary and economical significance, affecting dairy and beef cattle industries on a global scale. Comparative studies suggest that N. caninum consists of a globally dispersed, diverse population of lineages, distinguished by their geographical origin, broad host range, and phenotypic features. This viewpoint is however changing. While intraspecies diversity, and more specifically pathogenic variability, has been experimentally demonstrated in a myriad of studies, the underlying contributors and sources responsible for such diversity have remained nebulous. However, recent large-scale sequence and bioinformatics studies have aided in revealing intrinsic genetic differences distinguishing isolates of this species, that await further characterisation as causative links to virulence and pathogenicity. Furthermore, progress on N. caninum research as a non-model organism is hindered by a lack of robust, annotated genomic, transcriptomic, and proteomic data for the species, especially compared to other thoroughly studied Apicomplexa such as Toxoplasma gondii and Plasmodium species. This review explores the current body of knowledge on intra-species diversity within N. caninum. This includes the contribution of sequence variants in both coding and non-coding regions, the presence of genome polymorphic hotspots, and the identification of non-synonymous mutations. The implications of such diversity on important parasite phenotypes such as pathogenicity and population structure are also discussed. Lastly, the identification of potential virulence factors from both in-silico and next generation sequencing studies is examined, offering new insights into potential avenues for future research on neosporosis.

Detecting sequence variants in clinically important protozoan parasites.

Second and third generation sequencing methods are crucial for population genetic studies, and variant detection is a popular approach for exploiting this sequence data. While mini- and microsatellites are historically useful markers for studying important Protozoa such as Toxoplasma and Plasmodium spp., detecting non-repetitive variants such as those found in genes can be fundamental to investigating a pathogen's biology. These variants, namely single nucleotide polymorphisms and insertions and deletions, can help elucidate the genetic basis of an organism's pathogenicity, identify selective pressures, and resolve phylogenetic relationships. They also have the added benefit of possessing a comparatively low mutation rate, which contributes to their stability. However, there is a plethora of variant analysis tools with nuanced pipelines and conflicting recommendations for best practise, which can be confounding. This lack of standardisation means that variant analysis requires careful parameter optimisation, an understanding of its limitations, and the availability of high quality data. This review explores the value of variant detection when applied to non-model organisms such as clinically important protozoan pathogens. The limitations of current methods are discussed, including special considerations that require the end-users' attention to ensure that the results generated are reproducible, and the biological conclusions drawn are valid.

Annotating the 'hypothetical' in hypothetical proteins: In-silico analysis of uncharacterised proteins for the Apicomplexan parasite, Neospora caninum.

Neospora caninum is a parasite of veterinary and economic importance, affecting beef and dairy cattle industries globally. While this species has been recognised as a serious cause of disease in cattle and dogs for over 30 years, treatment and control options are still not available. Furthermore, whilst vaccination was identified as the most economic control strategy, vaccine discovery programs require new leads to investigate as vaccines. The current lack of gene annotation available for N. caninum, especially compared to the closely related model organism, Toxoplasma gondii, considerably hinders vaccine related research. Moreover, due to the high degree of similarity between the two organisms, a significant amount of gene annotation available for N. caninum stems from sequence homology between the species. However, there is a plethora of literature identifying conserved virulence factors between members of the Apicomplexa, which suggests that key players are contributing to successful parasite invasion, motility, and host cell attachment. In this study, bioinformatic approaches classified 125 uncharacterised proteins within the N. caninum genome, as transmembrane proteins with signal peptide sequences. Functional annotation assigned enriched gene ontologies for cell-adhesion, ATP binding, protein serine/threonine phosphatase complex, immune system process, antigen binding, and proteolysis. Additionally, 32 of these proteins were also identified as adhesins, or having adhesin-like properties, which were further characterised through the discovery of domains and gene ontology, to reveal their potential functional significance as virulence factors for N. caninum. This study identifies a new, small subset of proteins within N. caninum, that may be involved in host-cell interaction, parasite adhesion, and invasion, thereby implicating them as potential targets to exploit in the development of control options against the disease.

Genome Wide Identification of Mutational Hotspots in the Apicomplexan Parasite Neospora caninum and the Implications for Virulence.

Neospora caninum is an apicomplexan parasite responsible for neosporosis, a disease causing hind limb paralysis in dogs and abortion in cattle, resulting in substantial economic losses to beef and dairy industries. Marked differences in pathogenicity exist between N. caninum strains suggesting that intrinsic genetic differences exist between them. These differences likely exist in genes expressed during the tachyzoite lifecycle stage which is responsible for the pathogenesis of neosporosis. An improved understanding of these genetic differences is essential to understanding N. caninum virulence, though such knowledge is scarce. Using a variant detection workflow we compared the tachyzoite transcriptomes of two N. caninum strains with different virulence properties: NC-Liverpool (virulent) and NC-Nowra (avirulent). This workflow identified 3130 SNPs and 6123 indels between the strains, and nine markers capturing 30 variants were Sanger sequenced for both strains. Sequencing of these loci was extended to an additional eight strains and subsequent phylogenetic analysis supported a genetic population structure comprised of two major clades with no geographical segregation. Sequence polymorphisms within coding regions of tachyzoite-associated genes were concentrated on chromosomes XI and XII, with 19 distinct tachyzoite-associated SNP hotspot regions identified within coding regions of the N. caninum nuclear genome. The variants were predominantly located in loci associated with protein binding, protein-protein interactions, transcription, and translation. Furthermore, 468 nonsynonymous SNPs identified within protein-coding genes were associated with protein kinase activity, protein binding, protein phosphorylation, and proteolysis. This work may implicate these processes and the specific proteins involved as novel effectors of N. caninum tachyzoite virulence.