Field application of a novel multiplex qPCR assay reveals the occurrence of the zoonotic hookworm Ancylostoma braziliense in Nigerian dogs
Graphical abstract
Introduction
Dogs can harbour a plethora of intestinal parasites, some of which can also infect humans. A number of gastrointestinal parasites of veterinary or public health significance have been reported in Nigerian dogs including Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Dipylidium caninum, Taenia spp., and Trichiuris vulpis (Anene et al., 1996; Sowemimo, 2009; Mustapha et al., 2016; Idika et al., 2017; Moro & Abah, 2019). Of these parasites, Ancylostoma spp. were the most common parasites detected in pet dogs, with prevalence ranging from 14% to 70% (Anene et al., 1996; Sowemimo, 2009; Mustapha et al., 2016; Idika et al., 2017; Moro & Abah, 2019).
In addition to impacting canine health, the hookworms, Ancylostoma caninum, Ancylostoma braziliense, Ancylostoma ceylanicum, and U. stenocephala (Traub et al., 2004) are also zoonotic, therefore posing a risk to human health. Canine hookworms are well-known agents of cutaneous larva migrans in humans, with A. braziliense as the only species capable of causing “creeping eruptions”, which clinically manifest as prolonged, highly pruritic serpiginous lesions in the skin of human patients that may persist for over 100 days (Dove, 1932; Beaver, 1956; Brenner et al., 2003). Ancylostoma caninum is a well-known agent of eosinophilic enteritis and aphthous ileitis in humans (Croese et al., 1994; Walker et al., 1995; Prociv & Croese, 1996). Recently, the finding of A. caninum eggs in the faeces of human subjects has led to speculation that this parasite might have the potential to complete its life cycle in humans (Ngcamphalala et al., 2019; Furtado et al., 2020). Ancylostoma ceylanicum on the other hand, is commonly reported to cause patent infections in humans throughout the Asia Pacific, sometimes with accompanying clinical signs of diarrhoea and anaemia (Stracke, Jex, & Traub, 2020; Traub, 2013).
To date, there is a paucity of data on the occurrence of canine hookworms in Africa, with the majority of the surveys performed using morphological identification of eggs to differentiate between the species of hookworms. For instance, based on faecal floatation and Kato-Katz techniques A. caninum, A. braziliense and U. stenocephala have been reported in dogs from Nigeria (Abraham & Gloria, 2009; Nwoha & Ekwuruike, 2010; Okoye et al. 2011 Ayinmode et al. 2016; Moro & Abah, 2019). However, these techniques are not reliable given marked similarities in egg morphology of hookworm species, making the use of molecular tools fundamental to identify these parasites at species level (Lucio-Forster et al., 2012). PCR-based assays allowed the identification of A. caninum and A. braziliense from dogs in South Africa (Lamb et al., 2012; Ngcamphalala et al., 2019) and Kenya (Mulinge et al., 2020) and, of all the four species of canine hookworms in Tanzania (Merino-Tejedor et al., 2018).
A recently published Taq-Man based multiplex qPCR targeting the internal transcribed spacer 1 (ITS-1) region of the rDNA of all four canine hookworms was recently developed for the specific detection and discrimination of canine hookworm species in faecal samples (Massetti et al., 2020). The Taq-Man qPCR was field-validated for A. caninum, A. ceylanicum and U. stenocephala, but not for A. braziliense. In this study we characterised the species of hookworm infecting dogs in Nigeria through a novel high-throughput qPCR and report the occurrence of A. braziliense in the country.
Section snippets
Parasite material
A cross-sectional national survey of gastrointestinal parasites of dogs in Nigeria was conducted between November 2016 to December 2017 across the Nigerian Federal Capital Territory (FCT), Abuja in 11 states of Nigeria. Faecal samples were collected from the rectum of 885 dogs sourced from breeding kennels, abattoirs, and veterinary clinics and transferred into previously labelled screw cap containers. The faecal samples were kept on ice and transported to the Helminthology Laboratory of the
Results
The frequency and distribution of dogs positive for Ancylostoma spp. at microscopic evaluation (A) and for A. caninum (B), A. braziliense (C) and mixed species infections (D) by qPCR in Nigeria is shown in Fig. 1. Data on the distribution of dogs microscopy positive for hookworms in each region are listed in Table 1. Frequencies of occurrence of A. braziliense, A. caninum and mixed hookworm species infection are listed in Table 2.
Of the 885 animals, a total of 218 dogs (24.6%, 95% CI 21.8-27.6)
Discussion
In this study, we provide comprehensive information on the occurrence and species distribution of zoonotic hookworms infecting dogs in Nigeria. Further, we assessed the suitability of the newly developed high-throughput multiplex qPCR assays for the detection of A. braziliense for epidemiological investigations.
Based on classical parasitological methods, 24.6% of dogs from Nigeria were diagnosed with hookworm eggs in their faeces. Previous reports identified canine hookworms in 14-70% of dogs
Conclusion
This study demonstrates that A. braziliense is highly endemic in Nigeria and poses a potential zoonotic threat to locals and travellers. The multiplex qPCR was demonstrated to be a valid and accurate tool for the surveillance of hookworms on a large scale and for the diagnosis and characterization of A. braziliense under field conditions.
Conflict of interest statement
The authors have no financial, personal or professional interests that could be construed to have influenced the here presented work.
Data availability statement
All data are available within this manuscript.
CRediT authorship contribution statement
Luca Massetti: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Joshua Kamani: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Writing - review & editing. Anke Wiethoelter: Data curation, Formal analysis, Methodology, Supervision, Validation, Writing - review & editing. Phillip McDonagh: Funding acquisition, Supervision, Writing - review & editing. Vito
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Zoonotic hookworms of dogs and cats – lessons from the past to inform current knowledge and future directions of research
2021, International Journal for ParasitologyCitation Excerpt :Fast-forward a century, and the epidemiology and infection dynamics of A. ceylanicum infection in human hosts appears to have altered significantly. Since the advent of molecular diagnostic assays for species-specific detection of hookworm eggs in human (Traub et al., 2008; Papaiakovou et al., 2017; Hii et al., 2018) and animal stool (Traub et al., 2007; Massetti et al., 2021), A. ceylanicum has emerged as a predominant hookworm of dogs (Traub et al., 2007, 2014; Ngui et al., 2012; Ng-Nguyen et al., 2015; Fu et al., 2019; Zendejas-Heredia et al., 2021b) and the second most common hookworm infecting humans in the Asia Pacific region, after N. americanus (Table 1). Its distribution in humans largely mirrors its distribution in domestic dogs and cats (Traub, 2013; Stracke et al., 2020; Colella et al., 2021a).
Canine gastrointestinal parasites as a potential source of zoonotic infections in Nigeria: A nationwide survey
2021, Preventive Veterinary MedicineCitation Excerpt :The use of a novel multiplex qPCR (Massetti et al., 2020) on samples positive for hookworm-like eggs allowed us to resolve the difficulty associated with differentiation of hookworm species based on egg morphology. A. caninum and A. braziliense but not U. stenocephala were detected in Nigerian dogs (Massetti et al., 2021). Both of the hookworm species detected in this study are known to cause zoonotic infections in humans, with A. braziliense frequently causing cutaneous larva migrans syndrome (Bowman et al., 2010; Criado et al., 2012) and A. caninum potentially able to reach the adult stage and causing intestinal inflammation (Landmann and Prociv, 2003).
Conflict of interest statement
The authors have no financial, personal or professional interests that could be construed to have influenced the here presented work.
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Equal contribution