Introduction
Cystic echinococcosis (CE) is a parasitic zoonosis caused by cestodes. Regardless of the current knowledge on the genetic diversity, host range and the taxonomic challenges among certain members of the Echinococcus complex, it remains a neglected zoonotic disease across the world, and more so in Nigeria. Infection with this cestode results in serious veterinary and public health concerns (Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017) as economic losses are estimated to reach billions of US dollars annually (WHO, 2013). Echinococcus granulosus sensu stricto (s.s.) (G1,G3), is regarded to have the highest zoonotic potential, while other species in the complex include Echinococcus equinus (G4), Echinococcus ortleppi (G5), Echinococcus canadensis (G6–10) and Echinococcus felidis, with preference to different intermediate hosts (Lymbery, Reference Lymbery2017). Therefore, exploring the genetic variation within/between species and the species diversity in an endemic area is important to the control and management of CE. However, there are still contentious issues regarding the taxonomic status of the E. canadensis group (G6/7 G8/10) (Thompson, Reference Thompson2008; Lymbery et al., Reference Lymbery, Jenkins, Schurer and Thompson2015; Nakao et al., Reference Nakao, Lavikainen and Hoberg2015; Laurimäe et al., Reference Laurimäe, Kinkar and Moks2018).
In Nigeria, investigations have shown that the northern part of the country is endemic of CE. Yet, only recently was the G6/7 genotype reported to be majorly responsible for CE in animals in endemic communities (Ohiolei et al., Reference Ohiolei, Yan and Li2019a). In humans, CE is poorly investigated, although in recent times two unusual presentations have been reported: one is a case of musculoskeletal involvement with HIV coinfection in a patient admitted to The University of Jos Teaching Hospital in north-central Nigeria (Ozoilo et al., Reference Ozoilo, Iya, Kidmas, Uwumarogie and Hassan2007), and an orbital cystic echinococcosis manifested in protrusion of the eye and poor vision in the University College Hospital, Ibadan south-western Nigeria (Fasina & Ogun, Reference Fasina and Ogun2017). Nonetheless, it is believed that CE in humans is often misdiagnosed (Fasina & Ogun, Reference Fasina and Ogun2017), and the species/genotypes responsible for infection remain unknown. Here, we encountered and molecularly identified what we believed is the first record of the G1 genotype in a camel host from a northern Nigerian state, and highlight the potential public health importance.
Material and methods
Sample collection, polymerase chain reaction (PCR) amplification and sequencing
A fertile hydatid cyst of camel origin was collected in December 2018 from Sokoto modern abattoir, Sokoto state, Nigeria (13.0059°N, 5.2476°E) at postmortem during a routine inspection. Genomic DNA extraction was performed on a cut piece of the germinal layer using Qiagen DNeasy Blood and Tissue DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplification of the complete mitochondrial cox1 (1674 bp) (Kinkar et al., Reference Kinkar, Korhonen and Cai2019) and nad1 (894 bp) gene was achieved using previously described primer pairs (Wu et al., Reference Wu, Li and Zhu2018) in addition to a nad5 gene fragment (about 680 bp) (Kinkar et al., Reference Kinkar, Laurimäe and Acosta-Jamett2018a). PCR was conducted in 25 µl final volume using 2 mm MgCl2, 0.2 mm dNTPs, 5 µl 5× Taq buffer, 10 pmol of each primer, 0.5 µl Ex Taq DNA polymerase (5 U/μl, Takara), 0.5 µl of genomic DNA extract (~50 ng) and RNAse free water up to the final volume of 25 µl. The reaction cycled 35 times under the following conditions: denaturation at 95°C for 30 s, annealing at 55°C for 40 s and elongation at 72°C for 60–90 s after an initial denaturation at 95°C for 5 min, followed by a final extension at 72°C for 10 min.
Amplicons were visualized by electrophoresis in 1.5% (w/v) agarose gels in 1 × TAE (40 mm Tris-acetate, 2 mm EDTA, pH 8.5), stained with GelRed™, and viewed under UV light. The PCR products were purified using an Agarose Gel DNA Purification Kit (Axygen Biosciences, CA, USA), and then cloned to a pMD19 T-vector (Takara Bio, Japan) prior to sequencing (Beijing Tsingke Biotechnology Co., Ltd., China).
Identification and phylogenetic network analysis
Nucleotide sequences where viewed manually for correction of any nucleotide misread followed by alignment with retrieved sequences from Genbank in BioEdit v7.2.6 (Hall, Reference Hall1999). The identity of the isolate was confirmed via a nucleotide sequence BLAST search using the NCBI BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with G1 nucleotide sequences from other parts of the world. The median-joining network was inferred based on the sequences of the mitochondrial nad5 gene using PopART (http://popart.otago.ac.nz). Reference genotype G1 sequences for the network analysis were from Kinkar et al. (Reference Kinkar, Laurimäe and Acosta-Jamett2018b) (GenBank: MG672170–MG672220), while genotype G3 sequences were from Kinkar et al. (Reference Kinkar, Laurimäe and Balkaya2018c) (GenBank: MG682511–MG682544).
Results and discussion
The BLAST result of the mitochondrial sequences confirmed the identity of the isolate as G1 genotype with a 99–100% similarity with other G1 sequences deposited in GenBank. The median-joining network (fig. 1) based on the nad5 gene(680 bp) further distinguished the G1 genotype from G3 genotype. Meanwhile, A few nucleotide polymorphisms were observed between the Nigerian G1 isolate and a reference G1 sequence (GenBank accession: AB786664; host: human; origin: China) as follows: cox1 267A–267G; nad1 117T–117C; and nad5 355C–355T and 578T–578C. The G1 sequences from this study have been deposited in GenBank under the following accession numbers: MN199126 (cox1), MN199127 (nad5) and MN199128 (nad1).
Fig. 1. Median-joining network of the nad5 mitochondrial genes of a panel of 86 sequences consisting of Echinococcus granulosus sensu stricto representatives isolates/haplotypes from across the world, including our Nigerian isolate (haplotype H32). Reference genotype G1 sequences (51) = GenBank: MG672170–MG672220; genotype G3 sequences (34) = GenBank: MG682511–MG682544.
In West Africa, CE is largely under-investigated, unlike the northern and eastern sub-regions where data on CE epidemiology and the population genetic structure of the causative agents are available (Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017; Lymbery, Reference Lymbery2017). However, the few available data for the West Africa sub-region showed that E. canadensis G6/7 is basically responsible for CE in both humans and livestock (Mauti et al., Reference Mauti, Traoré, Crump, Zinsstag and Grimm2016; Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017; Ohiolei et al., Reference Ohiolei, Yan and Li2019a). Meanwhile, the role of camels as potential intermediate hosts for the G1 genotype has been documented in most African and Asian countries (Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017). Also, E. granulosus s.s., which consists of genotypes G1 and G3, is responsible for over 88.44% of global human CE burden (Alvarez Rojas et al., Reference Alvarez Rojas, Romig and Lightowlers2014). In Africa, E. granulosus s.s. and E. canadensis G6/7 are both responsible for the majority of CE infection in livestock and humans (Addy et al., Reference Addy, Alakonya and Wamae2012; Boufana et al., Reference Boufana, Lahmar, Rebai, Ben Safta, Jebabli, Ammar, Kachti, Aouadi and Craig2014; Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017), with cattle, sheep, goats and camels being massively involved in the transmission and maintenance of the infection (Ernest et al., Reference Ernest, Nonga, Kassuku and Kazwala2009; Boufana et al., Reference Boufana, Lahmar, Rebai, Ben Safta, Jebabli, Ammar, Kachti, Aouadi and Craig2014; Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017; Ohiolei et al., Reference Ohiolei, Yan and Li2019a), especially in poor pastoral communities with high stray dog populations and poor waste disposal systems in slaughterhouses (Ernest et al., Reference Ernest, Nonga, Kassuku and Kazwala2009; Omadang et al., Reference Omadang, Chamai, Othieno, Okwi, Inangolet, Ejobi, Oba and Ocaido2018).
In Nigeria, although the high prevalence of CE in livestock attracted a lot of concern between 1970 and 1990, not much has been done lately (see the review by Ohiolei et al., Reference Ohiolei, Yan, Li, Zhu, Muku, Wu and Jia2019b). In humans, the seemingly low CE prevalence has been attributed to poor surveillance, lack of differential diagnosis and poor knowledge by medical personnel (Fasina & Ogun, Reference Fasina and Ogun2017; Ohiolei et al., Reference Ohiolei, Yan, Li, Zhu, Muku, Wu and Jia2019b). In our previous study, where we reported the G6/7 genotype as being responsible for CE infection in livestock, the absence of the G1 genotype could have been a result of sample size limitation, as emphasized (Ohiolei et al., Reference Ohiolei, Yan and Li2019a). Nonetheless, the G1 genotype in the current study further suggests that other species/genotypes could potentially be present in the country and even within states. Furthermore, while transboundary animal movement through animal trade or smuggling could play a potential role in the distribution and prevalence of CE, the extent to which it influences the epidemiology in Nigeria remains unknown as most West African countries are largely uninvestigated (Deplazes et al., Reference Deplazes, Rinaldi and Alvarez Rojas2017; Ohiolei et al., Reference Ohiolei, Yan and Li2019a). However, infection with the G6 genotype in a Nigerien migrant was recently detected at a referral centre in Northern Italy. Based on the data, the authors suggested that the individual is potentially from an active transmission zone (Angheben et al., Reference Angheben, Mariconti, Degani, Gobbo, Palvarini, Gobbi, Brunetti and Tamarozzi2017). Interestingly, Sokoto, a state in the north-west zone of Nigeria where G6/7 genotype was previously reported (Ohiolei et al., Reference Ohiolei, Yan and Li2019a) and also the G1 genotype in the present study, borders Niger Republic to the north, indicating the possibility of transboundary CE transmission. Besides being a border state where unchecked animal movement could contribute to CE transmission, the state also shows other high-risk factors propagating CE transmission such as high frequency of stray dogs within the city metropolis and dog access to slaughterhouses. The city also hosts a significant number of households within the metropolis that keep dogs as pets that are most times allowed to roam freely, thus increasing the risk of human infection.
In conclusion, to the best of our knowledge, this study represents the first report of E. granulosus s.s. G1 genotype in Nigeria and the West Africa sub-region. With the previous report of the G6/7 genotype and the current detection of the genotype with the highest zoonotic potential, there is thus a need for heightened surveillance considering the public health significance of both genotypes. Furthermore, we recommend a massive screening of animal hosts alongside people living in endemic zones and human patients presenting symptoms suspected of CE in view of evaluating the burden of CE due to this genotype across the country and sub-region, as the previous absence of this genotype could be largely due to poor surveillance or a lack of molecular studies in the past. We also suggest future studies to consider the potential impact of animal movement across borders on the overall CE prevalence in the country.
Acknowledgements
We would like to thank the staff of Sokoto Central Abattoir for their assistance during sample collection, and Dr Etinosa O. Igbinosa, Head of Applied Microbial Processes and Environmental Health Research Group, University of Benin, Nigeria, for allowing us to access his laboratory facilities.
Financial support
This study was supported by the Central Public-Interest Scientific Institution Basal Research Fund (grant numbers 1610312017001, 1610312016012), the National Key Basic Research Programme (973 Programme) of China (grant number 2015CB150300) and the National Key Research and Development Plan (grant number 2017YFD0501301).
Conflicts of interest
None.
Ethical standards
Not applicable.
