key: cord-0259073-x6z28e50 authors: Bayegun, A. A.; Omitola, O. O.; Umunnakwe, C. U.; Akande, A. F.; Akinwale, O. P.; Mogaji, H. O.; Ademolu, K. O.; Gyang, P. V.; Odoemene, N. S.; Ekpo, U. F. title: Morphometric and molecular analysis of Schistosomes eggs recovered from human urines in communities along the shore-line of Oyan-dam in Ogun State, Nigeria date: 2022-04-16 journal: nan DOI: 10.1101/2022.04.11.22273687 sha: 7a33b2be461e7c859bd5eff5ecba36dad62b23a3 doc_id: 259073 cord_uid: x6z28e50 Background There are growing concerns that communities characterized with surface water, where both humans and livestock interact for agricultural, domestic, cultural, and recreational purposes, are likely to support hybridization between schistosome species infecting humans and livestock. This study therefore investigated the possible human infections with hybrid schistosomes in four schistosomiasis endemic communities along the banks of Oyan dam in Ogun State, Nigeria. Methods Human urine samples were collected in Imala-Odo, Abule-Titun, Apojula and Ibaro-Oyan communities. Recovered eggs were counted, photographed, and measured with IC Measure for Total Length, Maximum Width, and a ratio of egg shape. Eighty-seven unusual Schistosoma eggs shaped were molecularly characterised by PCR amplification of Schistosoma specific Dra1 gene. The amplicons were further subjected to PCR amplification of schistosome ITS-2 rDNA and right representative samples with varying gel band sizes were sequenced. Results A total of 1,984 Schistosoma eggs were analysed. The egg morphometrics were within the range of 70.90 - 262.30 {micro}m and 30.10 - 102.60 {micro}m for total length and width respectively. The length to width ratio was 1.60 - 4.06{micro}m. Majority of the eggs have the typical round-to-oval shaped eggs (1345, 67.8 %), followed by eggs with atypical spindle-shaped (639, 32.2 %) and eggs without spines (22, 1.1 %). Egg morphotypes were significantly different (p = 0.017). PCR amplification of Dra1 gene and ITS2 confirmed 54 (62.1%) of the eggs and 33 (61.1%) of Schistosoma origin. Sequencing of two of the DNA samples produced sequences similar to vertebrate S. magrebowiei (accession number UZAI01000474.1) and Asian S. japonicum (accession number SKCS01001458.1). Conclusion These findings suggest possibly that hybrids schistosome may be circulating in the human population in the study areas. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022 to study communities to obtain consents from the community leaders after explaining the 124 objectives of the research to them. Communities willing to participate in the study completed 125 written consent forms. Subsequently, parents and guardians/caregivers were also informed 126 about the study through community meetings organized by the consenting representatives. 127 Parents were asked to provide parental consents to their children by completing another 128 consent form, in addition to an assent form which was completed by children below 16 years 129 of age. Children whose parent did not agree to the study procedures were not recruited in the 130 research. promoted water contact activities such as bathing, swimming, drinking, washing clothes or 145 kitchen utensils and fetching of water from surface waterbodies, and more importantly shared 146 interaction between cattle with humans along the shoreline of the dam that surrounds these 147 communities (Fig 1) . Sample size determination and recruitment of study participants 151 The sample size for this study was determined using the formula described by [14] , 152 where n s is the sample size, p is the existing prevalence in the study 153 area, and d is the degree of accuracy. In determining the sample size, a prevalence of 47% [12], and a degree of accuracy of 5% was considered at 95% level of confidence. The preschoolers (1-4years), school-aged children (5-15 years) and those above 16-64 years 164 across study communities between October and November, 2019. Age of participants were 165 validated using birth-cards to avoid information bias. Three-hundred and eighty-four samples 166 were collected in dark, sterile 30ml universal containers and preserved with 70% ethanol. Collections were made between 10.00 and 14.00 hours as recommended [15] and transported . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.11.22273687 doi: medRxiv preprint to the laboratory in iceboxes. Urine samples were processed using sedimentation techniques, 169 and examined under the microscope for the presence of S. haematobium eggs. A total of 219 170 (57.0%) of the samples examined were positive for S. haematobium eggs, and seperated for 171 subsequent screening using morphometric and molecular methods. Bremen, Germany) computer software was used to measure the total length (including the 178 terminal spine) and the maximum width. The egg length/width ratio was subsequently 179 computed. Qualitative characteristics such as unusual morphology was noted, and the 180 presence or absence of the terminal spine was also recorded. The eggs were classified as 181 typical if they have a round-to-oval shape or atypical if they have spindle/elongated shape 182 [16, 17] . Putative hybrid schistosomes were identified by their atypical egg-shape [16, 18] . A 183 total of 639 (32.2%) eggs were characterized with atypical shape. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.11.22273687 doi: medRxiv preprint The mixture was centrifuged for 10 minutes at 13000 rpm, the aqueous layer of the mixture 194 was carefully removed into another 1.5ml reaction tube and 75 µl volume of phenol and 195 chloroform-isoamyl alcohol was added respectively. The mixture was pulse vortexed and 196 centrifuged for 10 minutes respectively and the aqueous layer was removed into another Table 1) . . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.11.22273687 doi: medRxiv preprint from Imala-Odo, Abule-titun, Apojola and Ibaro-oyan, respectively (Table 2) . Three egg 257 morphotypes were recorded in this study (Fig 3, 4 and 5) . The majority of the schistosome 258 eggs were of the typical round-to-oval shape (1345, 67.8%), and atypical elongated or (Table 3 ). The maximum width of the eggs was 102.60 µm with the minimum egg width of 275 30.10 µm and the mean egg width of 68.25 µm. Table 3 showed that the length/width ratio of 276 the eggs had a range of 1.60 µm -4.06 µm and the mean egg length/width ratio of 2.61 µm. 277 Figure 6 shows the scatterplot distribution of the egg length against the egg width which 278 represents an uneven distribution between the egg morphotypes from the areas. Only one 279 positive schistosome egg was recovered from the sampled cattle, and the egg length, width 280 and length/width was 330.10 µm, 139.50 µm and 2.40 respectively (Table 3) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.11.22273687 doi: medRxiv preprint (Table 4 ) (Fig 7) . Out of the 54 DNA samples, which were further subjected to PCR 289 amplification of the schistosome ITS-2 rDNA, thirty-three (61.1%) showed varying 290 differences in product sizes (Table 4 ) (Fig 8) 5, 7, 9, 11, 13-15 1: 1000bp marker, lanes 2, 6, 7, 9, 16, 17: no amplification, lanes 309 3-5, 8, 10-15, 18 and 19 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.11.22273687 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 16, 2022 Neglected Tropical Diseases in Sub-Saharan Africa: Review of 421 Their Prevalence, Distribution, and Disease Burden World Health Organization (WHO) guideline on control and elimination of human 424 schistosomiasis. 2022. Geneva: World Health Organization 426 3. World Health Organization (WHO). Schistosomiasis Detecting 469 hybridization in African schistosome species: does egg morphology complement 470 molecular species identification? Differences in the egg morphology and certain biological characteristics 472 of some African and Middle Eastern schistosomes, genus Schistosoma, with terminal-473 spined eggs African schistosomiasis in mainland China: risk of 518 transmission and countermeasures to tackle the risk. Parasites Vectors Hybridisation between the two major African schistosome species of humans High genetic variability of Schistosoma 388 We are grateful to the community leaders and residents of the study sites for their continuous 389 participation. Appreciation goes to the Neglected Tropical Diseases Department of the Ogun