Molecular characterization of South African Ehrlichia ruminantium field isolates and development of a qPCR assay for the evaluation of a multivalent inactivated vaccine

Heartwater is one of the major tick-borne diseases of both domestic and some wild ruminants in Africa and some Caribbean islands. The disease is caused by Ehrlichia ruminantium, a member of the phylum Proteobacteria and order Rickettsiales. Amblyomma genus ticks transmit E. ruminantium. Heartwater is a cause for concern, it challenges livestock farmers from introducing high producing exotic breeds to upgrade local stock. The exotic breeds are highly susceptible to E. ruminantium, and the mortality rate of susceptible animals ranges from 20 to 90% depending on the animal’s species, breed, and previous exposures. Ehrlichia ruminantium has diverse genotypes and immunogenicity caused by recombination that occurs between different isolates in the field. The diversity of E. ruminantium isolates render the commercially available vaccine ineffective against some virulent isolates. A development of an inactivated vaccine that can cross protect against virulent isolates is crucial. To develop an effective vaccine researchers must, isolate field E. ruminantium by in vitro culture system to provide the antigen required for the vaccine, characterize the isolates by genotyping to determine the possibility of their use in an inactivated cocktail vaccine. Then, the vaccine can be developed and tested. This study was therefore carried out to characterize South African E. ruminantium field isolates by Multi-Locus Sequence Typing (MLST) and develop a reverse transcription quantitative real-time polymerase chain reaction assay for the evaluation of a multivalent inactivated vaccine. A total of 1004 DNA samples were extracted from Amblyomma hebraeum ticks collected from cattle in Mpumalanga, Limpopo, and KwaZulu-Natal provinces of South Africa, and were tested for E. ruminantium by pCS20 Sol1 qPCR. The E. ruminantium positive samples were characterized by MLST using five housekeeping genes. Out of 1004 samples tested, overall E. ruminantium infection rate was 22% and provincially 19%, 22% and 27% in Mpumalanga, KwaZulu-Natal and Limpopo respectively. Sequencing of the housekeeping genes showed genetic variation in the sequenced regions and phylogenetic analysis revealed three main lineages, clade 1 made up of worldwide isolates (eastern, southern Africa, and Caribbean isolates); clade 2 comprised only West African isolates; and clade 3 consisted of the unique South African isolates, Omatjenne, Kümm2, and Riverside. MLST can clearly distinguish the South African genotypes from the distinct West African genotype. The genetic variation in the sequenced regions does not change the makeup and function of the protein encoded by the genes. In vitro culture using gut and salivary glands from E. ruminantium positive field ticks as an inoculum was initiated. First attempt of E. ruminantium in vitro culture, 19 days post inoculation, the cultures were E. ruminantium negative and at 25 days the cultures were contaminated. The second attempt had to be discontinued due to national lockdown caused by the worldwide corona virus pandemic. There is E. ruminantium positive tick material stored at -80 °C and that can be used in future to initiate in vitro culture of E. ruminantium. Ehrlichia ruminantium in vitro culture from tick material must be optimized and is a possible alternative for isolating field E. ruminantium without the need of infecting an animal. Also, the optimization of the method would make it easier to sequence genomes of E. ruminantium field isolates and fast track the development of an efficient vaccine. Methods to determine success of a developed vaccine need to be developed. One of the assays the current study developed was a reverse transcription quantitative real-time PCR (RT qPCR) assay to evaluate a multivalent inactivated vaccine against heartwater. This method requires selection and validation of housekeeping genes to obtain reliable results in immune markers for protection. Most housekeeping genes commonly used for qPCR analyses were selected from previous studies and the sheep homologues were selected from GenBank. The housekeeping genes were validated using Ct mean, CV of RPKM values and CT values and the ΔCt method to estimate the expression stability of each housekeeping gene. The genes were ranked for RT qPCR data normalization using an online tool, RefFinder. Immune markers for protection differentially expressed between surviving animals and animals that were treated (considered dead) were identified using RNA-seq data from a previous study. The most suitable choice combination of housekeeping genes that showed the least expression variation throughout the experimental conditions is SDHA, RPL22, YWHAZ and GAPDH. The immune markers for protection were identified and were upregulated in animals that survived heartwater challenge: CD41–ITGA2B, CD156A-ADAM8, CXCR5, CD8B, CD5, GZMB-LIKE, IFN-γ and TGFB1. Based on the developed RT qPCR the immune markers for protection were differentially expressed throughout the experimental conditions. The upregulation of the identified immune markers for protection does not inform protective immunity against the disease. In conclusion, the study revealed genetic diversity among E. ruminantium field isolates from A. hebraeum ticks in three provinces of South Africa. The RT qPCR assay developed to evaluate a multivalent inactivated vaccine against heartwater can be validated and applied in further studies. Also, there is a need for alternative genotypic genes that reveal immunogenicity of isolates for identifying best vaccine isolates for the development of an effective vaccine against heartwater. Optimization of E. ruminantium in vitro culture using tick salivary gland and gut is important.