Authors

I Owusu-Frimpong¹; EJ Tettevi¹; QD Quarshie¹; NA Kuma¹; N Imoro¹; Y Al-Mahroof¹; R Enchill¹; MK Ahiabu¹; MY Osei-Atweneboana¹;  ¹ Council for Scientific and Industrial Research Water Research Institute, Ghana

Discussion

Molecular xeno-monitoring has been a significant technique used to study the impact of vector-borne pathogenic diseases on humans and animals by employing disease surveillance in vector populations. It uses insects carrying pathogen genetic material as a non-invasive surrogate for infection in the human or animal population. In the case of onchocerciasis, the WHO has approved the O150 PCR in blackflies, a molecular xenomonitoring technique,  as part of the Onchocerciasis Elimination guideline. However, this technique is laborious and time-consuming, which could be influenced by human errors. Also, the high cost, non-availability, and delay of the ELISA component make the application of the O150 PCR difficult in resource-limited settings. Therefore, this study focused on developing a PCR-RFLP assay to detect and discriminate O. volvulus and O. ochengi in blackflies.
Bioinformatics analysis was employed to identify a unique restriction site within the COX1 mitochondrial gene sequences of O. volvulus and O. ochengi. HaeIII, unique to O. volvulus only, was identified as the restriction enzyme of choice for the discrimination. Onchocerca-genus primers were designed in the conserved regions of both O. volvulus and O. ochengi sequences to amplify a 650 bp fragment, which flunks the restriction site. From the conserved sequences, Onchocerca-COX1 probe was also designed to be used in the magnetic beads capture of Onchocerca-DNA from blackflies DNA pool. Assay validation was done with Onchocerca sp. sequence data retrieved from the NCBI Genbank. The wet-lab validation of this assay was performed with archived blackflies (S. damnosum sensu lato) collected in 2011 from Agbelekeme, an endemic onchocerciasis community. Triplicates of 50 and 100 Blackfly pools were performed separately for heads and bodies. Blackfly pool DNA was extracted, and the Onchocerca-DNA was captured with the Onchocerca-COX1 probe and magnetic beads. The PCR-RFLP assay was applied to the Onchocerca-captured samples, after which the PCR products were sequenced and analyzed.
The restriction site, GG|CC (HaeIII), was unique to only COX1 O. volvulus sequences in the NCBI GenBank since they produced 456 bp and 194 bp fragments from the 650 bp PCR product. Of the three 50 blackfly head pools, only 1 (1/3) carried infective O. ochengi larval stage. All three 100 blackfly head pools (3/3) carried infective O. volvulus larval stage. However, two of the three 100 blackfly body pools (2/3) were infected with O. ochengi. Only one of the two infected 50 blackfly body pools carried both O. volvulus and O. ochengi. The PCR amplicons with the restriction sites showed high homology with O. volvulus, whereas the unrestricted amplicons were highly homologous to O. ochengi after the DNA sequence analysis.
The novel PCR-RFLP assay has demonstrated its effectiveness in detecting and discriminating between O. volvulus and O. ochengi in blackflies. In addition, with zoonotic onchocerciasis in sight in some parts of the world, this tool will be useful for the early detection of potential zoonotic transmission of the bovine onchocerciasis in the human population, especially in Sub-Sahara Africa.