Elsevier

Acta Tropica

Volume 213, January 2021, 105758
Acta Tropica

Field application of a novel multiplex qPCR assay reveals the occurrence of the zoonotic hookworm Ancylostoma braziliense in Nigerian dogs

https://doi.org/10.1016/j.actatropica.2020.105758Get rights and content

Highlights

  • The novel qPCR allowed the detection of zoonotic hookworms in dogs from Nigeria

  • Ancylostoma braziliense is detected for the first time in dogs in Nigeria

  • Ancylostoma braziliense is endemic in Nigeria and poses a zoonotic threat

Abstract

A number of gastrointestinal parasites have been reported to infect dogs in Nigeria, some of which have zoonotic potential. Of these, hookworms are the most prevalent, with both Ancylostoma caninum and Uncinaria stenocephala reported in the country. In this study, we subjected 203 hookworm microscopy-positive samples of the 885 individual faecal samples collected from dogs in Nigeria to a recently developed multiplex qPCR for the detection and characterisation of canine hookworm species. The qPCR demonstrated a diagnostic sensitivity of 98% (95% CI 95-99.4) allowing the detection of A. caninum and A. braziliense in 81.3% (165/203, 95% CI 75.3-86.1) and 51.2% (104/203, 95% CI 44.4-58) of the microscopy-positive faecal samples of dogs from Nigeria, respectively and 34.5% (70/203, 95% CI 28.3-41.3) of mixed infections with both hookworm species.

The finding of A. braziliense is particularly worrisome given this is a well-known agent of persistent cutaneous larva migrans, commonly referred to as “creeping eruptions” in humans. Although this parasite has been diagnosed in locals and in people travelling in Nigeria suffering from dermatological illnesses, this represents the first molecular identification of A. braziliense in its canine reservoir in the country. These results update the occurrence and distribution of hookworm species affecting dogs in Nigeria highlighting the suitability of the newly developed multiplex qPCR assay as a high-throughput tool for the surveillance of zoonotic hookworms, globally.

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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    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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