The canine hookworm, Ancylostoma caninum (Nematoda: Strongylida: Ancylosmastoidea) (Figure 1), is the most prevalent and important intestinal nematode parasite of dogs in the USA, as well as in other parts of the world. A survey published in 1996 evaluating 6458 faecal samples from animal shelters across the USA yielded a prevalence for A. caninum of 19.19% (Blagburn et al, 1996). This was followed by a prevalence of only 2.5% from 1 199 293 faecal samples of pet dogs evaluated in 2006, with the prevalence depending on age, level of care and geographic location of the dog (Little et al, 2009). A study evaluating archived data of over 39 million canine faecal samples from 2012–2018 showed a stable prevalence of hookworm infections from 2012–2014 ranging between 1.5% and 2.3%. From 2015, the prevalence began to steadily rise each year, with an overall increase from 2015–2018 of 47% (Drake and Carey, 2019). This decrease in prevalence of more than 75% from 1996 to 2012–2014, even though samples from different populations of dogs were examined, could be largely because the awareness of anthelmintics for companion animals was very different than at the beginning of the 1990s. Additionally, a more frequent treatment to cover gastrointestinal nematodes was pushed by both veterinarians and parasitologists. However, in one large scale study evaluating the prevalence of intestinal parasites in dog parks throughout the USA, 7.1% of the samples were positive for hook-worms and of those, 98.2% were positive for A. caninum (Stafford et al, 2020). Interestingly, this prevalence is more than twice as high as that reported for 2018 by (Drake and Carey, 2019), and is more than 70% higher than the mean prevalence for 2017–2019 reported by Sweet et al (2021). There could have been some degree of underdiagnosing, thus underestimating, these prevalence rates because of technical factors (eg not all the eggs being detected at the faecal floatation because a centrifugal step was not used), or biological factors (eg different maturity of infections, as some could have been too early or too old to detect, and the females could be dying off as a result of senescence). However, taken together, these data suggest that hookworm prevalence is rapidly increasing, and that dogs that visit dog parks are at a higher risk of infection. Also, this shows that anthelmintics labelled for hookworms are showing not to be efficacious, as these pets are most likely to be on a monthly preventive for the canine heartworm, Dirofilaria immitis, and all these products have a drug with a label efficacy against hookworms. The high prevalence of hookworm found in the face of presumed anthelmintic use for heartworm could also be because of poor owner compliance, as demonstrated in European countries, where heartworm prevention should also be in place (McNamara et al, 2018).
Hookworms have evolved to be very successful parasites. Research has shown that infective larvae of Nippostrongylus brasiliensis, a hookworm of rodents, can evade the host's immune system through the secretion of a DNase II enzyme, and degrading the DNA backbones of the neutrophil extracellular traps (Bouchery et al, 2020). Furthermore, infection causes anaemia and diarrhoea.
There are three major anthelmintic drug classes currently available for treatment of hookworms in canines. The benzimidazoles (eg febantel and fenbendazole), are a class of anthelmintics whose mechanism of action is to bind tubulin, the eukaryotic cytoskeletal subunit of the microtubule, thus preventing the self-association of subunits onto the growing microtubules by the cell. Tubulin is a dimeric protein comprised of α and β monomers. Microtubules exist in a dynamic equilibrium with tubulin, with the ratio of dimeric tubulin to polymeric microtubules being controlled by a range of endogenous regulatory proteins and co-factors (Lacey, 1988; 1990; McKellar and Benchaoui, 1996). The first report of this mechanism was in Ascaris suum, when the normal microtubule matrix of intestinal cells was disintegrated after treatment with mebendazole (Borgers et al, 1975).
The avermectins and milbemycins (eg ivermectin, moxidectin, milbemycin oxime), are macrocyclic lactones that are used to control nematodes of human and veterinary importance (Campbell and Benz, 1984; Shoop et al, 1995). The mode of action is to selectively paralyse the parasite by increasing muscle permeability to Cl- ions in glutamate-gated chloride channels (GluCl) located in motor neurons (Cully et al, 1994). This mechanism paralyses the worm, interfering with pharyngeal pumping, motility and reproduction (Yates et al, 2003).
Tetrahydropyrimidines (eg pyrantel), are acetylcholine receptor-agonists which bind selectively to synaptic and extrasynaptic receptors on nematode muscle cells, and open Ca2+ ion channels. This forces contraction of the cell and induces a spastic paralysis in the worm (Martin, 1997). Intracellular recordings made with micropipettes from A. suum body muscles showed that pyrantel produces depolarisation, increased spike activity and muscle contraction (Aubry et al, 1970).
In registration studies for the Food and Drug Administration in the USA, febantel, moxidectin and milbemycin oxime all demonstrated efficacy of >99% (Food and Drug Administration, 1994; 2006; 2012). Fenbendazole demonstrated efficacy of >98% (Food and Drug Administration, 1983) and pyrantel demonstrated a somewhat variable efficacy, with a mean across studies of approximately 94%, where more than half of those studies yielded >99% (Food and Drug Administration, 1993).
Current management of hookworm infections in dogs
Typically, when a dog presents to a veterinarian with a faecal examination (Zajac et al, 2002), coproantigen-detection enzyme-linked immunosorbent assay test (Elsemore et al, 2017) or real-time polymerase chain reaction tests (Leutenegger et al, 2023a) positive for hookworms, the dog is treated with one or more drugs from the benzimidazole, macrocyclic lactone or tetrahy-dropyrimidine classes. If the dog then tests positive again in a future exam, the infection is attributed to reinfection or reactivation of encysted or arrested larvae (larval leak). Consequently, the same treatment regimen is often repeated, or the veterinarian may choose to use a drug from a different drug class. As a result, anthelmintic resistance is not diagnosed, and most often is not even considered as a likely cause of recurrent hookworm infections. Therefore, as resistance evolves and leads to more recurrent hookworm infections, veterinarians typically treat more often and rotate and/or combine drugs. However, they do not a perform faecal egg count reduction test to measure the efficacy of the various drugs administered. Thus, as long as one drug remains efficacious, the problem will appear to be managed, and recurrent infections will continue to be attributed to reinfection or reactivation of encysted or arrested larvae. However, once resistance to all drugs evolves, it is no longer possible to manage the infections, and the problem of anthelmintic resistance becomes more obvious.
In vitro tests are used to diagnose and measure levels of resistance by comparing the resistant isolate against a known susceptible isolate. Specifically in A. caninum, the only tests used have been the egg hatch assay (Jimenez Castro et al, 2019), larval feeding inhibition assay (Kopp et al, 2008a), larval migration inhibition assay (Kopp et al, 2008a), larval arrested morphology assay (Kopp et al, 2008a) and the larval development assay (Jimenez Castro et al, 2019; Leutenegger et al, 2023b).
Anthelmintic resistance in companion animals
The issue of whether resistance is likely to become a problem in parasites of companion animals has received relatively little attention, and when addressed, it has been viewed as an issue relating to the increased use of prophylactic helminth treatments in pets (Thompson, 2001), or not to be as common nor widespread as in large animals (von Samson-Himmelstjerna et al, 2021). Reports of anthelmintic resistance in parasites of small animals different from hookworms are very scarce. These include Toxocara canis (Dryden and Ridley, 1999), Dipylidium caninum (Jesudoss Chelladurai et al, 2018) and Dirofilaria immitis (Pulaski et al, 2014; Wolstenholme et al, 2015; Traversa et al, 2024). This is attributable to the lack of surveillance (ie measurement of treatment efficacy), indirect life cycles or the presence of paratenic hosts which increase the amount of refugia. This is mainly because of the different epidemiological factors that are present in the livestock situation but are not present in household pets, such as large numbers of animals in one place, high transmission rates and increased frequency of anthelmintic treatment.
However, the epidemiology of nematode transmission on greyhound farms much more closely resembles the epidemiological conditions present on livestock farms than the epidemiological conditions present in a pet home environment. Consequently, it would not be surprising if anthelmintic resistance were also to become a common problem on greyhound farms. Anthelmintic resistance in A. caninum was first reported in 1987 against pyrantel in a greyhound puppy that was imported to New Zealand from Australia (Jackson et al, 1987). Subsequently, several additional cases were diagnosed in Australia (Hopkins et al, 1988; Hopkins and Gyr, 1991; Kopp et al, 2007; 2008a; 2008b). However, there were no further cases of anthelmintic resistance reported in A. caninum between 2008 and 2019, when a report provided evidence of a case of resistance to benzimidazoles and macrocyclic lactones in an isolate of A. caninum obtained from a greyhound dog originating from Florida, USA in 2016 (Kitchen et al, 2019) and a report demonstrating resistance against all three of the most commonly used drug classes by in vitro, in vivo and molecular methods (Jimenez Castro et al, 2019). This was followed by a controlled efficacy study using an isolate that was originally obtained from a recently adopted retired racing greyhound dog in late 2017, where there was evidence of high levels of resistance to all classes of drugs approved for treatment of hookworm in dogs; fenbendazole, pyrantel pamoate and milbemycin oxime yielded efficacies of 26%, 23% and 9%, respectively. Emodepside, a drug of the cyclooctadepsipeptide class, demonstrated an efficacy of 99.6% and a 100% reduction in faecal egg count at 10 days post-treatment (Jimenez Castro et al, 2020).
The only mechanism of resistance currently known for anthelmintics is for the benzimidazole drugs. These drugs block the polymerisation of parasite microtubules by binding to the nematode β-tubulin protein monomers (Cleveland and Sullivan, 1985; Lacey, 1988; 1990). Briefly, single nucleotide polymorphisms in the isotype-1 of the β-tubulin gene family located at different codons have been found to be associated with benzimidazole resistance. Specifically for A. caninum, there have been reports of single nucleotide polymorphisms at:
Moreover, using faecal egg counts following anthelmintic treatment, researchers documented additional multiple anthelmintic drug-resistant A. caninum in 21 dogs of different non-greyhound breeds within the same kennel located in southeastern USA (Jiminez Castro et al, 2022). Interestingly, to address the origin of the multiple anthelmintic drug-resistant hookworms infecting the dogs in this kennel, using pre- and post-treatment hookworm eggs isolated from the fenbendazole treatment group, sequencing data revealed that these resistant worms contained the same two single nucleotide polymorphisms (in codon 167 and codon 134) that was recently reported in hookworms from greyhounds that originated from 16 different locations in eight different states. Thus, these genetic data indicate that the multiple anthelmintic drug-resistant worms in this kennel were almost certainly introduced by a dog that got infected elsewhere with worms that originally derived from a greyhound (Jimenez Castro et al, 2022). If the DNA extracted from A. caninum in a sample is positive for a fenbendazole resistance single nucleotide polymorphism, then either the detected parasites are homozygous or heterozogous for the allele which may be imparting phenotypic traits to the parasite, along with the resistance allele being passed along to the next generation (Jimenez Castro et al, 2021). Detection of drug-resistant A. caninum in dogs is a complex issue that deserves greater recognition before more multiple anthelmintic drug-resistant A. caninum infections are reported in an even wider geographic area and dog population distribution. This will demand a multifactorial approach to control this parasite, particularly in large commercial kennels where dogs are managed like livestock; however, it is the individual dog that transports encysted larvae and/or patent infections and can disseminate the multiple anthelmintic drug-resistant A. caninum to other geographic locations. This is why regular testing and follow-up to evaluate the efficacy of the anthelmintic is of paramount importance when persistent infections occur.
Public health concerns
Beyond the concerns for canine health, multiple anthelmintic drug-resistant in canine hookworms could present serious public health concerns because A. caninum is zoonotic. Humans infected percutaneously may develop cutaneous larva migrans (Leeming, 1966). Cases of eosinophilic enteritis (Prociv and Croese, 1996; Landmann and Prociv, 2003), folliculitis (Miller et al, 1991; Opie et al, 2003), localised myositis (Little et al, 1983), erythema multi-forme (Vaughan and English, 1998) and ophthalmological manifestations (Garcia et al, 2008), as well as patent infections have also been described.
Conclusions
Parasitic nematode genetic diversity is a constantly evolving scenario with resistant populations appearing as a result of treatment selection, random recombination and host migration. This produces a complex scenario of hard and soft selective sweeps. Based on haplotype data from A. caninum infections in the USA, there appears to be multiple origins of this resistance. Hence, faecal surveillance with affordable molecular tests with a rapid-turnaround are crucial to evaluate the molecular epidemiology of A. caninum infections to help gain insights iunto where these populations are emerging, the geographic distribution and evolution of resistant populations.