The prevalence of nematode infections of the free-roaming dogs in Digana and Pussellawa (93.2%) was comparable to the prevalence (76–97%) recorded in previous studies (e.g., Hantana, Peradeniya and Talawakelle) in the country [15–17, 24]. Sampled dogs were infected with both single (40.9%, n = 18 ) and mixed infections with two or more parasite species (52.3%, n = 23). Similar results have been obtained earlier by Perera et al., (2013) [15] and De Silva et al. (2022) [16], where the immune system is compromised in the individuals mentioned above with worm infections, thus providing a niche for other parasites to colonize.
Of the two study sites that were sampled, the overall prevalence of parasitic infections was higher in Digana (100%), particularly for all the nematode species that were common for both sites (i.e., Ancylostoma spp., Strongyloides sp., and T. canis), than in Pussellawa. This could be attributed to the difference in the soil microclimatic conditions (i.e., soil temperature and soil moisture) between the two study sites. The higher ambient soil temperature in Digana (28–31°C) may support the development of eggs and larvae, hatching of the eggs, facilitating better survival of infective eggs and larvae in soil and larval penetration of the hookworms [25], increasing the prevalence of STN infections. The lower soil temperature in Pussellawa (18–21°C) may decrease the prevalence of STN infections. Similarly, decreased soil temperature in Talawakelle (18–21°C) is known to substantially decrease the survival of plant nematodes decreasing the nematode diseases in tea plantations in the upcountry compared to the southern parts of the country (Dr. K.M. Mohotti, personal communication, February 12, 2021). Similarly, De Silva et al. (2022) [16] reported comparatively lower FECs in dog faeces in Talawakelle. Existing literature, for example, Okulewicz (2017) and Blum and Hotez (2018) [26, 27], also support the notion of the positive correlation between soil temperature and STN infection prevalence.
The prevalence of Strongyloides sp. in Digana (40.9%; n = 9) was significantly higher (Fisher’s exact test, p = 0.009) than that of Pussellawa (4.5%; n = 1). Similarly, FECs of Strongyloides sp. in Digana (8.015 (± 20.2) eggs per gram) were significantly higher (p = 0.006) than that of Pussellawa (0.455 (± 2.1) eggs per gram). The prevalence and intensity of infection in Strongyliodes sp. is higher in Digana than in Pussellawa. This could be explained by the life cycle of Strongyloides spp., as well as the differences in soil temperature and altitude of the sites. Eggs of Strongyloides spp. are rarely observed in faeces because they embryonate and hatch rapidly still inside the host; when the eggs are observed, they are embryonated eggs with the larvae inside. Therefore, rhabditiform larvae are mostly passed outside with faeces and passing eggs might indicate severe, life-threatening infections [28]. Strongyloides spp. has one of the most complex and unique life cycles, with an internal auto-infective and a restricted external free-living cycle. The internal auto-infective cycle is important for them to maintain constant contact with the host by migrating inside the host, ensuring their long-term survival independent of the external environment. However, external direct (homogenic) and indirect (heterogeneic) cycles are more dependent on the external environment conditions. During the external direct cycle, first-stage (L1) rhabditiform larvae or larvated eggs which later hatch into L1 larvae develop into infective filariform third-stage (L3) larvae, which are able to survive up to two weeks under favourable environmental conditions until they find a new host. In contrast, L1 rhabditiform larvae on soil develop into either free-living adult males or adult females which mates to produce larvae that can survive up to three weeks depending on the environmental conditions [28]. This external part of the life cycle of Strongyloides spp. possibly could increase the prevalence of the species in soil, which increases the infections and re-infections among their hosts, which explains the higher overall prevalence. The differences in STN prevalence and burdens observed between the two study sites can be directly related to the soil microclimatic conditions, due to the other sociodemographic indicators and level of urbanisation being similar in the study sites. Even though the two towns are located in the Kandy district, Digana falls under the intermediate zone (i.e., the zones found in the eastern and central regions of the country, receiving a mean annual rainfall between 1,750 mm and 2,500 mm). In contrast, Pussellawa falls under the wet zone (i.e., the zones found in the southwest region, receiving a mean annual rainfall of over 2,500 mm), which already explains the difference in their climates and altitudes. Therefore, overall variation in the prevalence, diversity, and FECs of STNs between the two sites can be related to abiotic factors such as annual temperature range, mean annual temperature, annual precipitation range and mean annual precipitation [29]. These factors contribute to the overall latitudinal diversity gradient (LDG) [30], indicating a prevalence of greater species richness and phylogenetic diversity in areas with ambient soil temperatures and soil moisture [31, 32]. Other local studies [16, 33] in the region noted a lower parasitic burden relative to what was expected and hypothesized that lower soil temperatures from their respective sampling sites were potentially responsible.
The most prevalent nematode species was Ancylostoma sp. (overall prevalence of 93.2%), Consistent with the studies mentioned above [15–17, 24]. This could be due to the production and shedding of a large number of eggs per week by an adult female worm [34, 35], contaminating the soil, thereby infecting more individuals. This was evident by the very high mean FECs of Ancylostoma sp. recorded in the current study from both Digana (Mean FEC = 113.833 (± 125.5) eggs per gram) and Pussellawa (Mean FEC = 195.015 (± 352.1) eggs per gram) areas. However, morphology and morphometry cannot be used to differentiate Ancylostoma and identify it up to the species level to determine which Ancylostoma species is more prevalent.
This study reports the first molecular identification of A. tubaeforme in dogs in Sri Lanka. In 2000, Dissanaike et al. [36] reported the recovery and identification of a dead adult A. tubaeforme female worm from an eye of a 46 years old man from Panadura, Sri Lanka. Although A. tubaeforme was reported to be endemic to the South Asian region (e.g., India, Thailand, Malaysia and Laos) [35–37], this is the first report of detecting A. tubaeforme in free-roaming dogs in Sri Lanka,. However, among the two Ancylostoma species reported here, A. caninum was the predominant dog hookworm species, consistent with De Silva et al. (2022) [16] findings. Despite having extracted the genomic material from a concentrated pellet of nematode eggs, the final yields from the PCR amplicons were potentially too low to yield a sequence for further phylogenetic analysis. It is assumed that this might be due to the relative rarity of A. tubaeforme eggs compared to A. caninum. However, the intensity and brightness of the agarose gel bands observed for samples containing A. tubaeforme lend credence to the presence of these species alongside A. caninum. A pairwise comparison of the sequence derived from the current study (AC1, Digana – Sri Lanka, GenBank accession number OQ101719) showed the highest similarity (99.17%) to one of the local sequences (GenBank accession no. MZ707153) from Talawakelle, Sri Lanka [16]. This high similarity between the two local sequences (i.e., OQ101719 (AC1) and MZ707153) suggests that the local variants could be more similar, compared to sequences derived from other regions of the world. Sequence AC1 derived from the current study clustered well with the sequences (i.e., KC755026, AM850106, LC054295 and LC177194) isolated from Asian countries with strong statistical support, illustrating the similarity of the A. caninum variant in Digana with the variants found in dogs from China, India and Laos and cats from China. Moreover, both A. caninum and A. ceylanicum clusters have both human and animal variants clustered together, suggesting the similarities between those variants, which might be indicative of the zoonotic potential of these Ancylostoma species.
