Genetic Diversity and Emerging Zoonotic Onchocerca Species in Human Populations in Taraba State, Nigeria

Background: A better understanding of parasite population genetic processes in speci�c biogeography is needed to support onchocerciasis elimination goals. The genetic diversity of Onchocerca micro�lariae was explored by amplifying a fragment of the 16S rRNA gene in the endemic area in Taraba State, Nigeria, Methods: Eight (8) communities were selected including six onchocerciasis endemic communities with records of ivermectin treatment having been annually distributed for 10 to 16 years, and two non-onchocerciasis endemic areas. The participants were 211 from endemic and 110 from non-endemic areas as control. Skin snips were taken from consenting participants by convenience sampling methods using a sterile sclera punch, from males and females residing within the communities for ten years and above or since birth, micro�laria and residual skin snips were preserved in RNALater® in a 1.5 ml micro-centrifuge tube. DNA was extracted from micro�lariae recovered and from those in residual skin snip specimens. Polymerase Chain Reaction (PCR) ampli�cation using speci�c primers for 16S genes was done to detect the identity of Onchocerca species. The ampli�ed products were sequenced and analyzed for species identity. Results: Multiple sequence alignment and phylogenetic analysis results showed distinct diversity of two sample sequences (G49_O.v. Gashaka and Y02_O.v. Yorro) from other samples from the study area and other regions, indicating emergence of a new polymorphic strain of O. volvulus. Report of a preliminary case of emerging zoonosis of O. ochengi infection in human (skin snips) sample (O. ochengi G44) in this study. Conclusions: It is clear there is genetic diversity of Onchocerca species and emerging zoonosis in the study site. We suggest further investigation on the extent and potentials of emerging zoonotic onchocerciasis by O. ochengi, in the light of cattle, Simulium vectors, environmental and humans overlap in the study area.


Background
Onchocerciasis is called river blindness because the blood-feeding black ies that transmit the ultimately blinding disease are prevalent throughout many major river basins in Africa in lush, fertile land alongside the rivers in which they breed [1].
It is estimated that 120 million people are worldwide are at risk of onchocerciasis.Numbers of people infected with Onchocerca volvulus are 26 million, with dermal micro lariae in 31 sub-Saharan African countries [2].
Nigeria ranks among those countries with the highest burden of the disease in the world [2], accounting for about a third of the global prevalence with estimated 8 million Nigerians infected with O. volvulus.Nigeria has about 50 million persons in 40,000 communities at risk with over 6.5 million suffering from severe itching or dermatitis, 500,000 are blind [3].
The annual treatment regimen with ivermectin has achieved a high degree of onchocerciasis control in some areas [4,5].Several eld studies have shown the reduction in the transmission of infection [6].However, in some parts of Nigeria there are still places where active transmission continues and where the prevalence of O. volvulus is till high [7].
About one-third of the Onchocerca lariidae are in Africa, where the Simulium damnosum sibling species are not the only the main vectors of O. volvulus but also those species that transmit the cattle parasite Onchocerca ochengi and Onchocerca ramachandrini, a parasite of warthogs [8].A reliable and rapid taxonomic identi cation of parasitic disease agent is essential for their correct diagnosis; hence molecular detection of species diversity has become increasingly popular [9].The close similarity of Onchocerca species necessitates the exact identi cation of the developmental stages as a condition for assessing transmission indices in endemic areas [10].
In Northern Cameroon, which borders Taraba State, Nigeria, the prevalence of micro lariae of O. ochengi in cattle can be as high as 66 -71% [11,12].However, since O. ochengi is closely related to O. volvulus, the routinely used O-150-target, commonly used as a diagnostic marker for O. volvulus clusters with other Onchocerca species and therefore discrimination is problematic [8].
However, molecular phylogenetic analysis of three ribosomal DNA gene fragments [12S, 16S rRNA genes, and NADH dehydrogenase subunit 5 (ND5)] from ve Onchocerca species in Mali has shown potentials for use in species separation and segregation in diversity studies [13].Also, the development of DNA probes speci c for O. volvulus and O. ochengi have become an additional tool in population genetics studies and for discriminating among Onchocerca species speci cally [14,15].Almost all vectors of human onchocerciasis are also partially zoophilic.Thus they can transmit infective larvae of animal Onchocerca species from cattle, warthogs and antelopes [16,17] and other zoonotic species.Onchocerca volvulus populations may misdiagnose during surveillance studies affecting the accuracy of epidemiological data and improper interpretation of human onchocerciasis [18].
In a multi-site study evaluating ve Community Directed Treatment with Ivermectin (CDTI) projects in Nigeria and Cameroon, over one-quarter of the age-eligible people in the study communities were low complaints and may act as a reservoir for continued transmission of onchocerciasis [19,20].
Thus, the pressure on the parasite due to over two decades of Mass Drug Administration (MDA) regime of Ivermectin distribution in this endemic area and the potential co-transmission of human and animal lariae presents confounding issues regarding emerging species diversity are the key ndings in this article.

Study Area
Out of a total of 8 communities selected in 4 Local Government Areas of Taraba State, six were onchocerciasis endemic while two were from non-onchocerciasis endemic communities (Figure 1)..The study area is characteristic of southern Guinea Savanna with annual mean temperatures ranging between 27 to 37°C, relative humidity between 62 -97%) and an average rainfall of 180mm.The physical relief, which surrounds the study area are mountains about 1000m in height [7].The onchocerciasis endemic focus extends across the international border to Northern Cameroon.The area is innervated with rivers and tributaries of streams traversing the communities and draining into the river Benue, which provide numerous breeding sites for the black ies [21] Ethical clearance and Participants consent Ethical approvalfrom ABU Zaria Nigeria, Committee for Human Subject Research (ABUCUHSR/Zoology/2020/006).Oral informed consent was obtained from the participants.

Skin snip collection
Super cial skin biopsies ("skin snips") were taken from around the iliac crestas previously described by [19], following Rapid Epidemiological Assessment (REA) guidelines [22].Each snip from individual participants was immediately placed into the 1.5ml microcentrifuge tubes with approximately 100 μl physiological saline and incubated for 12-24 hours [23].The emerged micro lariae were examined microscopically at 40-400× magni cation.Then, the micro lariae and the corresponding residual skin snips were transferred to a 1.5ml Eppendorf tube containing 200 μl of RNAlater® preservative [24] and stored at -20°C until used for PCR ampli cation.

DNA Extraction, PCR Ampli cation, Puri cation and Sequencing
Genomic DNA was extracted from skin biopsies samples using the QIAamp Investigator kit (Qiagen, Germany).The molecular identi cation of Onchocerca species was done by ampli cation of ribosomal DNA (rDNA) targeting a fragment of the 16S gene, with speci c sets of primers: FW-primer: 5´-TGGCAGCCTTAGCGTGATG-3´, RV-primer: 5´-CAAGATAAACCGCTCTGTCTCAC-3´ [15].The PCR mix was in a total reaction volume of 25 μl consisting 0.5 μl MgCl2, 0.5 μl dNTP, 0.5 μl forward and reverse primer, 2.5 μl Green Dream Taq buffer, 0.5 μl Taq polymerase and 1 μl of DNA template.The thermocycler conditions were calibrated to run an initial denaturation for 4 min at 94˚C, denaturation at 94˚C for 30 s, annealing at 52.5˚C for 30s and elongation at 72 ˚C for 90 s for 35 cycles and a nal elongation of 7 min at 72 ˚C and held at a temperature of 4ºC overnight till further analyses.
The PCR products were resolved in 3% TBE agarose gel electrophoresis process, stained with Stain G (SERVA Electrophoresis, Germany) for 1 hour.Amplicons were visualized in a gel documentation system consisting of UV Benchtop Imaging System, Transilluminator (Hero®, Germany) and the images photographed and saved as JPEG formats.The sequencing was achieved when the amplicons in the agarose gels were excised and puri ed using the GeneJET Gel extraction and puri cation kit as described by the manufacturer's instructions (Thermo Scienti c, Dreieich, Germany).Sanger sequencing was carried out by the Microsynth Sequencing Laboratories in Göttingen, Germany.The sequenced samples were analysed using Geneious and Basic Local Alignment Search Tools (BLAST) algorithms similarity search of16S rRNA Onchocerca species in the GenBank in the BLAST (http://www.ncbi.nlm.nih.gov/BLAST).Partial DNA sequence of 16S genes of the samples was edited in Bioedit (http://www.mbio.ncsu.edu/BioEdit) and further aligned with sequences retrieved from published 16S sequences of O. volvulus, O. ochengi, O. siisa, O. gutturosa, and O. gibsoni as well as published O. volvulus from other regions and an out-group sequences in the GenBank for comparison and validation of inter and intra-speci c variability [25] in MEGA X (http://www.megasoftware.net)algorithms using neighbour-joining consensus trees construction [26].

Results
Species identi cation and sequence characteristics Table 1 and 2 show the reference sequences (16S) derived from Mali, Yemen, Cameroon, Ghana, Uganda and the sample sequences from the study area.The pairwise nucleotide distance in the p-distance model showed no p-distance (0.00 %) between a sample in this study (Y07_O.volvulus Nigeria) with those from Cameroon, Mali, Yemen, Uganda and Ghana (Table 1).However, there was pairwise-distance between two samples in this study (Y02_O.v.Yorro and G49_O.v._Gashaka), 3.00% and 7.00% respectively with O. volvulus sequences obtained from some countries (Table 1).

Phylogeny of the genus Onchocerca
Sequence data from the study and GenBank of some in-group species O. volvulus, O. ochengi 'Siisa', O. ochengi, O. gibsoni, O. gutturosa and O. ramachandrini and an outgroup species D. immitis obtained from GenBank andplaced as basal species of the genus with high bootstrap support and their relationship.Cluster 1 in Figure 3 is the closeness of the sequence data of O. volvulus in some countries (Cameroon, Uganda, Mali and Ghana) from GenBank to the sample (O.volvulus_Y07_Yorro_Nigeria) in this study.
There was diversity observed in two samples sequences in cluster 2 indicated in black words to O. volvulus sequence from some countries obtained from the GenBank (Fig 3).The species in cluster 3 in red words is O. ochengi identi ed in this study (Fig 3).
The tree show the Onchocerca evolutionary diversity in this study and other members of in-group species of some countries from the GenBank.Clusters: 1 in green colour, 2 in black colour and 3 in red colour.
The tree showed the Onchocerca evolutionary diversity in this study and some in-group species sequences obtained from the GenBank indication in colours: blue = O. volvulus species diversity identi ed; green = similarity of sample of study site and O. volvulus obtained from GenBank; red = similarity of sample of study site and O. ochengi obtained from GenBank; black = other in-group species obtained from GenBank.

Discussion
This study showed the presence of micro lariae (9 of 211 persons examined by skin-snips; 2.47%) as observed by previous parasitological examination carried out by other studies in the same study area [7,21,27,28].The molecular identi cation of Onchocerca species in this study is much simpler methods needed for diagnosis of Onchocerca species [29].
We observed some genetic diversity within the populations of O. volvulus.Two samples (Y02_O.v.Yorro and G49_O.v._Gashaka) showed a diversity of 3.0% and 7.0% respectively (Table 1) in the p-distance model, suggesting the review of generalized diagnostic test assay for transmission assessment based on the peculiarity of the endemic areas.The pairwise nucleotide diversity (Table 2) obtained from GenBank as presented in this study con rms the submission by [30] and [25] that O. ochengi and O. Siisa are much closer to each other -indeed, they freely interbreed and thus belong to one species [25] than to O. volvulus [29].
Another noteworthy nding of our study are the two samples (G49_O.v. Gashaka and Y02_O.v.Yorro) that showed distinct variation (diversity) if compared to those from other regions, as indicated in Single Nucleotide Polymorphism (Figure .2).This might point towards the emergence of a new strain of O. volvulus in our study area.No previous similar work has been carried out in this region to con rm this claim.So we suggest further investigation is needed to verify this nding.This work suggests the likelihood of a diverse sister species emerging nearer to O. volvulus than to O.ochengi or O. siisa [25].These may be due to pressure of the drug and systematic non-compliance to ivermectin in this endemic area known for continual transmission despite nearly two decades of Mass Drug Administration of Ivermectin [7,28] which might be leading to changes or genetic drift among the parasite populations in the study area.
The study recorded a preliminary novel incidence of O. ochengi (Figure 3) in the human skin biopsies.The patient is a healthy 48 years female farmer that was born and residing in Kwaitap village in Gashaka LGA, Taraba State, Nigeria.A punch of skin snip was obtained from the upper hip (buttock)of the patient, who have no nodule neither any clinical manifestation associated with lariasis, the study site is characterized with abundance of cattle populations that cohabitate and graze among the human population.The status of animal onchocerciasis is still unknown in Nigeria even though there is a high cattle population in human onchocerciasis endemic areas who serve as reservoirs for bovine onchocerciasis (O.ochengi); almost all vectors of human onchocerciasis are zoophilic, thus transmitting both animal and human Onchocerca species might likely occur [31] Although intensive studies have been carried out in Cameroon [18], there is no report of O. ochengi being isolated from human populations yet.Nonetheless, in Japan [32], zoonotic lariasis caused by O. dewitteijaponica has been described in a resident of Hiroshima Prefecture, Honshu, Japan and Onchocerca lupi has been reported to infect dogs, cats, and humans [33].It is against this background that this study is proposing further investigation on the threat and potentials of emerging zoonotic onchocerciasis (O.ochengi) due to cattle, Simulium vectors, and humans overlap in the study area.

Conclusion
The study revealed a novel incidence of O. ochengi (G44_O.o.Gashaka) in human populations.Two samples (G49_O.v. Gashaka and Y02_O.v.Yorro) have distinct variation (diversity) from those of other regions, suggesting an emergence of new polymorphic strains of O. volvulus associated with the persistent transmission of onchocerciasis in long term treatment with ivermectin.The study is relevant in future application of the drug regime and dynamics of the epidemiology of the disease.The application of molecular tools in this study has shown their potential in the detection during mapping and other dynamics epidemiology and control strategies.

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Figure 1 Map
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Figure 4 A
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