Hookworm infection affects millions globally, leading to chronic conditions like malnutrition and anaemia. Among the hookworm species, Ancylostoma ceylanicum stands out as a generalist, capable of infecting various hosts, including humans, cats, dogs and hamsters. Surprisingly, it cannot establish in mice, despite their close phylogenetic relationship to hamsters. The present study investigated the development of A. ceylanicum in immunodeficient NSG mice to determine the contribution of the immune system to host restriction. The infections became patent on day 19 post-infection (PI) and exhibited elevated egg production which lasted for at least 160 days PI. Infective A. ceylanicum larvae reared from eggs released by infected NSG mice were infectious to hamsters and capable of reproduction, indicating that the adults in the NSG mice were producing viable offspring. In contrast, A. ceylanicum showed limited development in outbred Swiss Webster mice. Furthermore, the closely related canine hookworm Ancylostoma caninum was unable to infect and develop in NSG mice, indicating that different mechanisms may determine host specificity even in closely related species. This is the first report of any hookworm species completing its life cycle in a mouse and implicate the immune system in determining host specificity in A. ceylanicum.

Soil-transmitted nematodes (STNs) place a tremendous burden on health and economics worldwide with an estimate of at least 1.5 billion people, or 24% of the population, being infected with at least 1 STN globally. Children and pregnant women carry the heavier pathological burden, and disease caused by the blood-feeding worm in the intestine can result in anaemia and delays in physical and intellectual development. These parasites are capable of infecting and reproducing in various host species, but what determines host specificity remains unanswered. Identifying the molecular determinants of host specificity would provide a crucial breakthrough towards understanding the biology of parasitism and could provide attractive targets for intervention. To investigate specificity mechanisms, members of the hookworm genus Ancylostoma provide a powerful system as they range from strict specialists to generalists. Using transcriptomics, differentially expressed genes (DEGs) in permissive (hamster) and non-permissive (mouse) hosts at different early time points during infection with A. ceylanicum were examined. Analysis of the data has identified unique immune responses in mice, as well as potential permissive signals in hamsters. Specifically, immune pathways associated with resistance to infection are upregulated in the non-permissive host, providing a possible protection mechanism that is absent in the permissive host. Furthermore, unique signatures of host specificity that may inform the parasite that it has invaded a permissive host were identified. These data provide novel insight into the tissue-specific gene expression differences between permissive and non-permissive hosts in response to hookworm infection.

Hookworms are some of the most widespread of the soil-transmitted helminths (STH) with an estimated 438.9 million people infected. Until relatively recently Ancylostoma ceylanicum was regarded as a rare cause of hookworm infection in humans, with little public health relevance. However, recent advances in molecular diagnostics have revealed a much higher prevalence of this zoonotic hookworm than previously thought, particularly in Asia. This study examined the prevalence of STH and A. ceylanicum in the municipalities of Palapag and Laoang in the Philippines utilizing real-time polymerase chain reaction (PCR) on stool samples previously collected as part of a cross-sectional survey of schistosomiasis japonica. Prevalence of hookworm in humans was high with 52.8% (n = 228/432) individuals positive for any hookworm, 34.5% (n = 149/432) infected with Necator americanus, and 29.6% (n = 128/432) with Ancylostoma spp; of these, 34 were PCR-positive for A. ceylanicum. Considering dogs, 12 (n = 33) were PCR-positive for A. ceylanicum. This is the first study to utilize molecular diagnostics to identify A. ceylanicum in the Philippines with both humans and dogs infected. Control and elimination of this zoonotic hookworm will require a multifaceted approach including chemotherapy of humans, identification of animal reservoirs, improvements in health infrastructure, and health education to help prevent infection.

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue®, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100–1 μg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screening.

The relationships between the number of infective larvae (L3s) to which animals were exposed and the establishment of A. ceylanicum and N. americanus in hamsters were examined. There was no evidence of density-dependent constraints on the establishment of A. ceylanicum in the range 10–1000 larvae, but an experiment in which the range was extended to 1500 L3s gave a significant negative correlation between the magnitude of the infecting dose and percentage establishment. Even so the percentage reduction was relatively small, approximating to 0·89% per 100 larvae administered, and in practice density-dependent constraints on establishment are unlikely to affect experiments with this species in which much lower doses (<250 L3) are generally employed. The range of doses for N. americanus L3s was smaller (10–400). Of the four experiments reported, two gave a significant reduction of establishment with increasing dose and two did not. When the data was split into low doses (<100 L3s) and high doses (>100 L3s), falling establishment with increasing dose was only detected in the lower dose range. There was no difference in the establishment when doses of 100 L3s were compared with 250 or 400 L3s. On balance, it was concluded that density-dependent constraints on establishment of N. americanus in hamsters were not marked and would have little significant effect on experiments utilizing fewer than 200 L3s (approximately 7·6% reduction between 10 and 200 L3s). These results are discussed in relation to host regulation of hookworm burdens.

Ancylostoma ceylanicum and Nippostrongylus brasiliensis decarboxylated most of the amino acids examined, but only a few at signficant rates. The former nematode in general possessed higher activites. Striking differences between the two parasites were, however, noticed regarding the metabolism of some of the amino acids. For instance, while alanine followed by asparate produced highest amounts of 14CO2in the presence of A. ceylanicum, proline exhibited maximum decarboxlation in case of N. brasiliensis. Tyrosine, on the other hand, did not libreate detectable CO2with either parasite. Likewise, although large number of amino acids underwent transmination with 2-oxoglutarate, only some of them elicited appreciable activity for any two parasites.

The immune response of hamsters to a chronic hookworm infection has been investigated. Ancylostoma ceylanicum caused long term infections in hamsters which were associated with prominent changes in secondary lymphoid organs. The mesenteric lymph nodes and spleens increased rapidly in size stabilizing at approximately 3–4 times the weight in control animals by weeks 3–7. Cells from both the mesenteric lymph node and spleen, after an initial period of increased blast cell activity, became less reactive in the latter stages of infection. Serum antibody responses were marked, commencing in weeks 3–4 and increasing in intensity throughout the 10 week period of measurement. The results are discussed in relation to their contribution to the understanding of human hookworm infection.

Laboratory bred DSN hamsters were exposed to varying doses of infective larvae of Ancylostoma ceylanicum (orally) or Necator americanus (percutaneously) and were autopsied at times which corresponded to a period immediately before cessation of growth of worms or soon afterwards. A total of 829 (404 male and 425 female) A. ceylanicum and 1582 (781 male and 801 female) N. americanus were measured. At worm burdens of fewer than 100, the length of A. ceylanicum appeared to increase with infection intensity and no evidence was found that growth was retarded under crowded conditions. In an experiment comparing directly low (mean worm burden = 22) and heavy infections (mean worm burden = 180) significant negative associations between both weight and width, and worm burden were detected, but length again increased with worm burden. In contrast, 5 experiments with N. americanus indicated negative relationships between measures of worm size (length, width, wet and dry weight) and worm burden. It was concluded that N. americanus is subject to regulation by density-dependent processes within the host while A. ceylanicum is not sensitive to the same degree.

The course of primary infection with a hamster-adapted strain of Ancylostoma ceylanicum was studied in inbred DSN and randomly bred WO/GD and WO/CR hamsters. Infective larvae were administered orally and began to develop in the small intestine without embarking on a tissue migration. Only the occasional larva was detected in other organ sites. It was concluded that the developing larvae moulted on days 3–4 and again to the pre-adult stage about 9–11 days post-infection. Worm burdens in infected hamsters were stable for at least 11 weeks after infection. There was no sudden expulsive phase and some adult worms survived for over 200 days. Overall the sex ratio of worms in groups of hamsters killed concurrently was about 50% although occasionally the ratio was biased in favour of one sex in individual animals. The blood packed cell volume (PCV) was significantly depressed 2 weeks following infection and continued to decline until a point of stability was achieved 4–5 weeks post-infection. The PCV subsequently remained depressed throughout the period of observation. Infected hamsters lost weight if kept in groups, but not when housed in separate cages. Groups of animals which lost weight did not recover to normal values within 11 weeks of infection. It is suggested that this model of hookworm infection has scope for exploring aspects of the host-parasite relationship which the canine models cannot fulfill adequately.

Trickle infections with a hamster-adapted strain of the hookworm Ancylostoma ceylanicum were studied by administering doses of 5–30 larvae twice weekly to inbred DSN hamsters. The worm burdens were regulated at very low levels, and this was found to be independent of the size of the infective dose. It is likely that there is some turnover of adult worms during trickle infection, as larvae of all stages were recovered from most time-points. This regulation is immunologically mediated and both the worm burdens and fecundity were increased in hamsters that were immunosuppressed by cortisone treatment. A trickle infection regime also induced a 67% protective immunity to a single subsequent challenge. The resistance that occurred during trickle infections was, however, incomplete, and some worms were found to survive in hamsters that had been repeatedly infected for over 10 weeks. Thus, although hamsters are capable of regulating the infection, some worms are capable of surviving the host immune effectors.

The feeding behaviour of parasitic 3rd-stage larvae (L3) of the hookworms Ancylostoma caninum, A. ceylanicum and Necator americanus was examined. Less than 11% of A. caninum L3 recovered from the small intestines of dogs infected orally were feeding at 4–48 h post-infection (p.i.). and none of the A. ceylanicum L3 recovered from the intestines of orally infected hamsters had resumed feeding. All L4 of both species recovered at 36 and 48 h p.i. had resumed feeding. On the other hand, approximately 16% of the A. ceylanicum L3 recovered from the skin of percutaneously infected hamsters at 18 h were feeding, and the percentage feeding increased to nearly 58% at 44 h p.i. Necator americanus L3 recovered from the skin of percutaneously infected neonatal hamsters resumed feeding at 6–12 h p.i. and reached 90–94% by 18 h. Feeding began to decline at 66 h, and reached 29% at 120 h p.i. This decrease was associated with the migration of larvae from the skin to the lungs. By 192 h p.i. over 95% of the larvae had reached the small intestine, and all had moulted to the L4. The results indicate that parasitic L3 resume feeding in the skin during percutaneous infections, and suggest that feeding by hookworm L3 correlates with the resumption of development.