Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae.

Napier grass Stunt Disease (NSD) is a severe disease of Napier grass (Pennisetum purpureum) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants. Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12,5 µg of fresh plant material.

Wildebeest-associated malignant catarrhal fever: perspectives for integrated control of a lymphoproliferative disease of cattle in sub-Saharan Africa.

Wildebeest-associated malignant catarrhal fever (WA-MCF), an acute lymphoproliferative disease of cattle caused by alcelaphine herpesvirus 1 (AlHV-1), remains a significant constraint to cattle production in nomadic pastoralist systems in eastern and southern Africa. The transmission of WA-MCF is dependent on the presence of the wildlife reservoir, i.e. wildebeest, belonging to the species Connochaetes taurinus and Connochaetes gnou; hence, the distribution of WA-MCF is largely restricted to Kenya, Tanzania and the Republic of South Africa, where wildebeest are present. WA-MCF is analogous to sheep-associated MCF (SA-MCF) in many aspects, with the latter having sheep as its reservoir host and a more global distribution, mainly in developed countries with intensive livestock production systems. However, unlike SA-MCF, the geographic seclusion of WA-MCF may have contributed to an apparent neglect in research efforts aimed at increased biological understanding and control of the disease. This review aims to highlight the importance of WA-MCF and the need for intensified research towards measures for its integrated control. We discuss current knowledge on transmission and geographical distribution in eastern and southern Africa and the burden of WA-MCF in affected vulnerable pastoral communities in Africa. Recent findings towards vaccine development and pertinent knowledge gaps for future research efforts on WA-MCF are also considered. Finally, integrated control of WA-MCF based on a logical three-pronged framework is proposed, contextualizing vaccine development, next-generation diagnostics, and diversity studies targeted to the viral pathogen and cattle hosts.