Infection and Immunity

Main objectives of the project
The project aims to unravel virulence of Campylobacter fetus (C. fetus) subspecies, to identify biomarkers for diagnostic applications and to study the presence of C. fetus in cattle in Indonesia.

Scientific approach and research methodology
This project consists of three parts which are: i) the study on virulence factors of C. fetus in cell lines or organoids, ii) identification of subspecies differences from genome comparisons, and iii) a field study in Indonesia to investigate carriage of C. fetus subspecies. The scientific approaches and research methodology of these three parts are outlined below.

To have a more comprehensive view on C. fetus virulence, we will use both phenotypic and genotypic approaches to understand Campylobacter fetus fetus (Cff) and Campylobacter fetus venerealis (Cfv) host specificity and invasiveness. For phenotypic analysis, we will use fluorescent-tagged genetically modified Cff and Cfv strains with various cell lines and organoids as an infection model to study the interaction (attachment and invasion). The Cff and Cft mutant strains contain GFP on the pRYEL1 vector plasmid that would indicate their location in specific cell lines and organoids, visualised by expression of the green fluorescein protein. Together with RNAseq analysis, this in vitro experiment can provide answers on the host specificity of C. fetus and its capability to invade eukaryotic cells.
From the genotypic aspect, we will do molecular analyses for both Cff and Cfv chromosomal and extra-chromosomal DNA using bioinformatics tools for virulence genes mapping and exploring putative pathogenesis-related genes. In a previous study, some conserved and also inconsistent variation among C. fetus subspecies was shown. Genetic variation in genes or even single-nucleotide variation is scattered on both genomes. We will identify these regions by assembling C. fetus DNA into complete genomes using long (Nanopore), and short (Illumina) reads to determine which part is responsible for subspecies diversity. The use of long reads will enable an investigation of highly repetitive regions such as the surface antigen protein (SAP) locus. The findings would be fundamental for the next step, diagnostic tools development. We will start our study investigating the differences in the sap genes because of the importance of the SAP locus as a surface layer for antigenic variation and resistance against the host immune system.
To extend our knowledge on Bovine Genital Campylobacteriosis (BGC) in Indonesia, we will perform a field study in a region that lacks a surveillance system for C. fetus. We assume that Cff and Cfv might play an essential role as causative agents for fertility problems in cattle. The insufficient continuous monitoring system at the national level and import of live cattle may put Indonesia at risk for spread of BCG. It is imperative to find out the carriage rate of C. fetus in Indonesia as it may result in substantial economic losses due to infertility and sporadic abortion due to BGC. In this project, we will perform strain isolation and molecular characterization of Cff and Cfv from cattle in Indonesia and estimation of prevalence. The strains from this study will be compared with strains from other countries in the global database available in Utrecht.

Expected scientific and societal impact
The scientific and societal impact that could be attained through this project are:

We will get a better understanding of host-Campylobacter interaction and its tropism. Our project findings will fill the scientific gap and help scientist and practitioners to get a better view of the pathogenesis of Campylobacter since the research and publications in this particular topic are relatively rare.
The result of our bioinformatics study is necessary for researchers to develop molecular diagnostic tools for Campylobacter subspecies detection. The DNA fragments or genes that are responsible for Campylobacter subspecies diversification are still unknown. Moreover, there is no single reliable diagnostic method that could be applied for that purpose nowadays.
The lack of knowledge and capacity on detecting campylobacteriosis is considered as primary shortages for Indonesia. As a nation that is aiming for a national self-sufficient beef production program as stated by the government in 2014, Indonesia has to have sufficient knowledgeable and skilled experts in beef production and health, including the infertility problems that might be caused by campylobacteriosis. The study of Campylobacter in Indonesia will result in a positive impact, specifically for local scientists and decision-makers in terms of knowledge and capacity. This project will involve some researchers and veterinarians in Indonesia. Our goal is that they will learn some essential techniques in sampling, isolation, identification, and epidemiological analysis of campylobacteriosis in cattle in Indonesia.

Expected PhD thesis outline (Chapters)
The outline of the PhD thesis in experimental chapters papers:

In vitro study of Campylobacter fetus infection in cell lines or organoids The paper will demonstrate host-cells and etiologic agent interaction, specifically for Cff and Cfv. It will answer the host target specificity among Campylobacter fetus subspecies.
Bioinformatics analysis of SAP locus of Campylobacter fetus, SAP recombination and strategy for evading the immune system;
The distribution of virulence genes on genomic DNA and plasmids and their contribution for pathogenesis;
Description of putative genes for virulence factors and persistency.
Prevalence and distribution of Campylobacter fetus in indigenous and non-indigenous cattle in Indonesia
Phenotypic and genotypic characterization of Cff and Cfv in Indonesia for analyzing genetic distance and phylogenetic trees compare with the other strains all over the world.

Contact
Sidna Artanto