This project was approved by the National System of Conserved Areas of Costa Rica (R-SINAC-ACG-003-2021). Costa Rica is in Central America within the equatorial tropical region. Among its 5 million residents, 766,000 are school-age children. Coyotes (Canis latrans) live around national parks and are widely distributed across the plains, mountains, forests, and tropics. To investigate the prevalence of hookworm and threadworm infections among coyotes, during May–October 2021, we collected and tested coyote scat from the Central Conservation Area in central and the Guanacaste Conservation Area in northwestern Costa Rica (10). To confirm coyotes were the origin of the collected fecal samples, we cross-referenced data from a separate study conducted on the same samples that employed restriction fragment-length polymorphism–based methods and metabarcoding analyses of the vertebrate 12S ribosomal RNA gene (11).

We extracted genomic DNA from fecal samples (n = 111) using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, https://www.qiagen.com) according to manufacturer instructions and added an extra initial bead-beating step. We performed 2 TaqMan (Thermo Fisher Scientific; https://www.thermofisher.com) probe–based real-time quantitative PCRs in duplicate to detect all present canine hookworm species (A. ceylanicum, A. caninum, A. braziliense, and Uncinaria stenocephala) and Strongyloides spp. threadworms, as described elsewhere (10,12). We subjected samples positive for A. ceylanicum to molecular characterization of a partial region of the cox1 gene (13). We downloaded nucleotide cox1 sequences from dogs, humans, and cats from GenBank to assess the phylogenetic relationship among Ancylostoma spp. parasites and hosts.

We used MEGA11 (https://www.megasoftware.net) for aligning sequences and determining the best-fit substitution model. We conducted phylogenetic analyses using MrBayes version 3.2.7a (https://nbisweden.github.io/MrBayes/download.html) for Bayesian and RAxML version 8.2.12 (https://github.com/stamatak/standard-RAxML) for maximum-likelihood inference. We annotated the resulting phylogenic tree using TreeViewer version 1.17.6 (https://treeviewer.org) and constructed a haplotype network using PopArt (https://github.com/jessicawleigh/popart).

Of the 111 samples collected, 103 had sufficient feces for successfully extracting DNA, determined by amplifying a housekeeping gene of canids as DNA extraction control. Of those 103 samples, 59 (57.3%, 95% CI 46.6%–62.2%) harbored >1 zoonotic hookworms; A. caninum accounted for most infections (53.4%; 95% CI 43.3%–63.3%), followed by U. stenocephala (4.7%, 95% CI 1.5%–10.7%), and A. ceylanicum (2.8%, 95% CI 0.56%–8.05%). Strongyloides spp. threadworms were found in 3.8% (95% CI 1.04%–9.3%) of coyotes.

Figure 1

Figure 1. Phylogeny of Ancylostoma ceylanicum hookworms from coyotes in from Guanacaste Conservation Area, Costa Rica, 2021, and reference sequences. Gene tree shows a partial region of the mitochondrial ...

Figure 2

Figure 2. Median joining haplotype network showing global Ancylostoma ceylanicumsequences for cats, humans, and dogs and coyotes from Guanacaste Conservation Area, Costa Rica, 2021 (dashed box). Each circle represents a...

We obtained a 289-bp amplicon showing 99.6%–100% nucleotide identity with A. ceylanicum sequences from 2 positive A. ceylanicum samples (GenBank accession numbers OR801542 and OR801543). We determined phylogeny from 40 sequences that included A. ceylanicum sequences from dogs, cats, and humans, and U. stenocephala and A. caninum sequences from dogs. Phylogenetic analyses enabled separation of A. ceylanicum nucleotide sequences from coyote samples from Costa Rica from A. caninum and U. stenocephala sequences (Figure 1). Upon examining the median joining network of global A. ceylanicum sequences, we identified 5 distinct clusters, 4 of which were shared between domestic animals and humans and only 1 with dogs. We observed no clear population structure among countries represented by an admixture of haplotypes. A. ceylanicum haplotype from coyotes was unique but separated by only a few mutational steps from those from dogs and humans, and from zoonotic clusters (Figure 2).

We report zoonotic hookworm infections in coyotes from Costa Rica. Our data provide further evidence of the endemicity and local transmission of A. ceylanicum hookworm in the Americas and show that coyotes might play a role in transmitting this parasite to other animals. Even though coyotes are widely distributed across North and Central America and are established reservoirs of other zoonotic hookworms, such as A. caninum and U. stenocephala (14), their role as hosts of A. ceylanicum hookworm remains unexplored. Furthermore, detection of A. ceylanicum hookworms in wildlife poses questions about the parasite’s possible role as a disease agent in endangered animals, such as jaguars, giant anteaters, and Baird’s tapir, that live in sympatry in the Guanacaste Conservation Area (15).

Costa Rica has effectively controlled soil-transmitted helminths in humans, and residents no longer require preventive chemotherapy. However, the Pan American Health Organization still recommends continuous monitoring and evaluation to maintain control of soil-transmitted helminths (15). Although eliminating the need for preventive chemotherapy constituted a great milestone, doing so leaves untreated persons vulnerable to infection with zoonotic helminths, including A. ceylanicum. We therefore advocate for increased surveillance of A. ceylanicum hookworm in domestic animals, wildlife, and humans in the Americas to monitor and prevent transmission of zoonotic hookworms. Expanded surveillance is crucial considering active transmission of A. ceylanicum hookworm in humans has been reported in Ecuador, another country listed by the Pan American Health Organization as no longer requiring preventive chemotherapy (5,15). In-depth population genetics studies are needed to assess whether increases in the reports of A. ceylanicum hookworms in the Americas are attributable to recent human and animal migration from areas where the parasite is highly endemic. More robust epidemiologic data on A. ceylanicum infection in domestic animals and humans would help public health agencies assess the extent of local transmission in Costa Rica and other regions of the Americas and the role of animals in transmitting the parasite to humans.