Status of Anthelmintic Resistance against Ancylostoma spp. in Naturally Infected Dogs

The most common infectious agents of dogs are gastrointestinal parasites. In India, the most common intestinal nematode parasite of dogs is Ancylostoma caninum, also known as the canine hookworm. The pathophysiology linked to hookworm infection includes iron-deficiency anaemia, hypoalbuminemia and enteritis. These conditions are followed by diarrhoea that may contain fresh or digested blood. A. caninum causes traumatic lesions by biting the intestinal mucosa. Feeding sites bleed continuously for a long time before the worm relocates and causes normocytic hypochromic anaemia (Qadir et al., 2011). Furthermore, by producing cutaneous larva migrans, eosinophilic enteritis and patent infections in humans, these parasites may also represent a serious threat to public health (Jimenez Castro et al., 2020).
       
Thus, to protect dogs from zoonotic illnesses and to guarantee their health, regular parasitological surveillance and suitable treatment approaches are needed (Miro et al., 2007). In developing nations such as India, pyrantel, ivermectins and benzimidazoles are frequently used singly or in combination to treat various parasitic infections. Benzimidazoles work by preventing tubulin from polymerizing into microtubules. Pyrantel Pamoate produces an excitatory blockade by functioning as an agonist at the nicotinic acetylcholine receptor (nAChR), which depolarises neuromuscular blocking. This causes a massive overstimulation of the target nematode’s muscle, which in turn causes persistent spastic contraction and paralysis. Ivermectin works by preventing the transmission of parasite nerves linked to GABA, which paralyses the pharyngeal muscles and causes the worm to become flaccidly paralysed.
       
Multi-drug resistant (MDR) nematode populations are on the rise globally, as a result of total dependence on anthelmintic medications in ruminants and their heavy usage for worm management (Kalpan, 2004). In contrast, anthelmintic resistance in A. caninum has developed far more slowly, with only a few examples reported. Jackson et al., (1987) reported the first case of pyrantel resistance in a greyhound puppy imported from Australia; this was followed by many cases (Hopkins et al., 1988; Hopkins and Gyr, 1991; Kopp et al., 2007; Kopp et al., 2008a and 2008b). According to recent reports, a greyhound dog’s strain of A. caninum was resistant to benzimidazoles and macrocyclic lactones (Kitchen et al., 2019). Soon after, Jimenez Castro  et al. (2019) reported multiple drug resistance to all three of the main drug classes that were most frequently used to treat the parasite in dogs.
       
There are fewer field surveys on the efficacy of anthelmintics in dogs and cats than in ruminants. Therefore the present study was planned with two objectives, to estimate the prevalence of gastrointestinal parasites in dogs in Rewa, India and to study the therapeutic efficacy of fenbendazole, pyrantel and ivermectin against gastrointestinal nematodes in dogs.

Study area
 
Rewa is situated at 24^o31'57"N latitude and 81^o17'32"  E longitudinal at 309 MSL (Mean sea level), having mild generally subtropical climate with average rainfall of 1128 mm.
 
Faecal sample collection and examination
 
Faecal samples of 754 dogs of different age, sex and breed were randomly collected. Signalment of these dogs and consistency of these faecal samples were also recorded. These samples were examined for parasitic infections by standard qualitative and quantitative methods (Zajac et al., 2021). The prevalence data were analysed using the chi square test (Snedecor and Cochran, 1994).
 
Efficacy of anthelmintics
 
40 dogs with more than 300 eggs per gram of faeces were chosen based on the prevalence study and placed into four groups, each with 10 dogs. Group G1 was kept as undewormed control and Group G2 was given fenbendazole @ 50 mg/kg BW, Group G3 was given pyrantel @ 15 mg/kg BW and group G4 was given ivermectin @0.6 mg/kg BW orally. Each animal rectum was sampled for faeces on day 0 (pre-treatment), day 10 and day 14 after treatment. Using the RESO (version 2.0) computer program (Martin and Wursthorn, 1991), the EPG data were statistically analysed in accordance with the recommendations provided by the World Association for the Advancement of Veterinary Parasitology for the detection of anthelmintic resistance (Coles et al., 1992; Geurden et al., 2022). If the FECR was less than 95% and the lower 95% confidence limit was less than 90%, resistance to anthelmintics was taken into consideration. Resistance was suspected if only one of these two conditions was satisfied.

169 of the 754 faecal samples were positive i.e. the overall prevalence of gastrointestinal parasites was 22.41% (169/754). This value is in agreement with 27.08% prevalence reported previously by Qadir et al., (2012) in dogs in Jabalpur (M.P.). This prevalence was comparatively lower than 65.5% reported by Shahat et al., (2022). According to Kumar et al., (2015), dogs from Junagadh, Gujarat, have an overall prevalence of gastrointestinal parasites of 21.97%. In Palampur, Himachal Pradesh, Moudgil et al., (2016) also found that the general prevalence of gastrointestinal parasites in dogs was 28.04%. One possible explanation for the lesser prevalence of gastrointestinal parasites in the present study could be the higher number of domestic dogs (n =611) compared to stray dogs (n =143). The prevalence was comparatively higher in stray dogs (32.87%) as compared to that of pet dogs (19.97%). Pet owners might be more aware of the deworming of their pets.
       
Parasite wise prevalence was significantly highest for Ancylostoma spp. 12.33 per cent (93/754) followed by that of Toxascaris leonina 6.23 per cent (47/754), Dipylidium caninum 5.44 per cent (41/754), Toxocara canis 2.79 per cent (21/754), Isospora spp. 2.12 per cent (16/754) and Spirometra spp. 0.13 per cent (1/754). Kurumadas et al., (2020), Duncan et al., (2020) and Sukupayo and Tamang (2023) also reported highest prevalence of Ancylostoma spp. in dogs. Out of 169 positive samples, 45 samples (26.63%) were positive for mixed infection while 124 samples (73.37%) were positive for mono infection i.e. only one parasite species was present (Table 1). The results corroborate those of Mukaratirwa and Singh (2010) and Palmer et al., (2008), who found that the percentage prevalence of mono infection was higher than that of mixed infection. The prevalence was only slightly higher in adult (23.43%) than young dogs (21.94%) contrary to other studies (Merga and Sibhat, 2015; Moudgil et al., 2016) that found puppies to be more infected than adults. There was no discernible difference in the prevalence of gastrointestinal parasites between the two age groups in the present study. The findings, however, are consistent with those of Stafford et al., (2020), who found that young adult dogs (45.6%) had a greater prevalence of gastrointestinal parasites than immature adults (20.4%) and puppies (2.4%). In females, the prevalence was non-significantly higher (22.97%) as compared to that of male dogs (22.08). The increased frequency of gastrointestinal parasites in female dogs may be caused by a stress factor linked to their physiology, which lowers their immunity to parasite infection. Mirzaei and Fooladi (2012) reported non-significant difference in prevalence percentage between male and female dogs i.e. 7.7 and 6.5 respectively, while Satyal et al., (2013) reported non-significantly higher prevalence of gastrointestinal parasites in female dogs (51.6%) as compared to that of male dogs (39.8%). Merga and Sibhat (2015) reported that sex has no significant association with parasite infection in dogs. The monsoon season’s prevalence (23.02%) was not statistically different from that of the summer season (21.66%), (Table 1). Lower prevalence in summer season might be due to adverse climate conditions resulting in delay in infectiveness of parasite eggs. In a retrospective study, Vatsya et al., (2010) also found that dogs had a higher frequency of gastrointestinal parasites during the rainy season than during the summer.


       
In the current investigation, Ancylostoma spp. was the most common parasite (12.33%). Ancylostoma has a high pathogeneity for pups, which can lead to oxidative stress, anaemia, hypoalbuminaemia, impaired immunity and a diminished response to vaccinations, in addition to its several modes of transmission. Young puppies’ limited iron reserves make them particularly vulnerable. When this parasite causes an acute infection, puppies may die. Given that benzimidazole is frequently used either alone or in conjunction with pyrantel and/or praziqantel for canine deworming. During the first three months of their lives, puppies were routinely dewormed every 2-3 weeks and after that, on a regular basis (Ridley et al., 1994). Regular deworming can put more pressure on the parasite to select for anthelmintic resistance. The pharmaceutical companies create and promote use of endo-parasiticides that can combat a wide range of intestinal parasites in dogs.  However some researchers have shown in a few different parts of the world, routine, metaphylactic use of broad-spectrum anthelmintic combinations may foster the emergence of anthelmintic resistance (AR) as compared  to deworming for a specific parasite (Jackson et al., 1987;  Kopp et al., 2007, 2008a).
       
According to Kopp et al., (2007), the FECRT has limited usefulness as a quantitative measure of anthelmintic efficacy against A. caninum isolates of intermediate resistance status, but it may help identify highly resistant or susceptible isolates. FECRT is a screening tool that can be used to find resistant isolates that require further investigation.  In the present study, efficacy of fenbendazole at day 10th post treatment was 96% with lower 95% confidence interval of 88% (Table 2). Efficacy of pyrantel at day 10 post treatment was 97% with lower 95% confidence interval of 88%. Efficacy of Ivermectin at day 10 post treatment was 99% with lower 95% confidence interval of 93%. All the three anthelmintic were 100% effective at day 14 post treatment (Table 2). These results showed that Ancylostoma worms were susceptible for ivermectin while suspected resistance was shown against fenbendazole and pyrantel.


       
Studies on livestock nematodes have revealed that SNPs in the β-tubulin isotype 1 gene produce benzimidazole resistance. Tyrosine is substituted for phenylalanine (TTC®TAC) by SNPs in codons 200 and 167, whereas alanine is substituted for glutamate (GAG®GCG) by an SNP in codon 198. It has been discovered that all three of these SNPs independently confer resistance, despite the fact that the 200 and 167 SNPs are more frequently linked to resistance (Dixit et al., 2017). The β-tubulin protein undergoes structural alterations due to these amino acid mutations, which prevents the BZs from binding. However, the canine nematode, Ancylostoma caninum, seems to have a distinct mechanism (Albonico et al., 2004b; Schwenkenbecher et al., 2007; Diawara et al., 2013a). The canine hookworm A. caninum strain Aca-KGR is resistant to multiple drugs (Kitchen et al., 2019). The β-tubulin isotype 1 gene’s codon 167 in this strain contains an SNP that converts the amino acid phenylalanine to tyrosine. This first multidrug-resistant strain of A. caninum promises anthelmintic resistance can develop in natural populations of hookworms as well. Even while widespread AR in canine intestinal helminths is not present at this time, further scientific data is clearly needed immediately.

In dogs, Ancylostoma spp. was the most common parasite. The Ancylostoma worms were found susceptible to Ivermectin but the status of resistance in fenbendazole and pyrantel was found to be suspected. Consequently, molecular research is needed to verify the resistance status of hook worms in the dogs residing in the area. Additionally, it will aid in comprehending the mechanism of resistance to anthelmintics.

The authors thank the Dean of the College of Veterinary Science and A.H., Rewa, for providing the resources needed for this study.

The authors declare no potential conflicts of interest with respect to research, authorship and/or publication of this article.