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Background: Hundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration., Methodology/principal Findings: In this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group., Conclusions/significance: These results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms., Competing Interests: The authors have declared that no competing interests exist.

Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.

A 26-year-old male member of the Australian Defense Force presented with a history of central abdominal pain of 4 weeks duration and peripheral eosinophilia consistent with eosinophilic enteritis. Acute hookworm disease was diagnosed as the cause. Adult worms recovered from feces after therapy with albendazole were morphologically consistent with Ancylostoma ceylanicum. As the patient had been deployed with the Regional Assistance Mission to Solomon Islands for 6 months prior to this presentation, it is very likely that the A. ceylanicum was acquired in Solomon Islands. Until now, it has been assumed that any Ancylostoma spp. recovered from humans in Solomon Islands is A. duodenale. However, this case demonstrates that human hookworm infection acquired in the Solomon Islands could be caused by A. ceylanicum., Competing Interests: We have no conflict of interest related to this work.

A 14-month-old, male American Bulldog presented to Texas A&M University Veterinary Medical Teaching Hospital in August of 2012 for anorexia, hydrophobia and gradually worsening neurologic signs. Grossly hemorrhage on the left side of the caudal cerebrum and cerebellum was observed and histologically corresponded with necrohemorrhagic and lymphoplasmacytic encephalitis associated with adult nematodes. Based on morphology and molecular analysis, these were identified as Ancylostoma sp., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hookworm infections as well as other intestinal nematodiases are endemic in China. In this case, a 70-year-old male showed symptoms of chest tightness, shortness of breath, and both lower extremities edema. The diagnostic result was chronic renal insufficiency, chronic kidney disease (5th stage), and renal anemia at first. Then, he received treatment with traditional drugs. However, this treatment did not help to alleviate the symptoms of the patient significantly. The results of gastroendoscopy showed hookworms in the duodenum, also confirmed by pathology examination. Anemia was markedly ameliorated after eliminating the parasites. The results mentioned above suggested that ancylostomiasis was the leading causes of anemia in this patient, and the etiology of anemia in uremic patients should be systematically considered. Especially when anemia could not be cured by regular treatments, rare diseases should be investigated.

This study compared the course of Ancylostoma ceylanicum infection in hamsters infected with different inocula and the consequences for the host and helminth populations. The average of adult worms recovered, according to the number of third stage larva used, were 28.0, 24.8, 24.6, and 24.8% to inocula size of 25 L3, 75 L3, 125 L3, and 250 L3, respectively. The size of the inoculum did not affect the establishment, survival, or fecundity of adult helminths. Reductions in the red blood cell and hemoglobin levels in the infected group were inversely proportional to the number of white blood cells. Moreover, differential cell counting revealed a positive correlation between the worm load and leucocyte numbers. The humoral response against excretion-secretion antigens was more robust and sensitive compared with the response against crude extract, with no direct linear correlation with the number of worms. The effect of the population density was more evident in females.

We report a case of Ancylostoma ceylanicum infection detected by endoscopy. It was diagnosed and confirmed using polymerase chain reaction (PCR) and DNA sequencing. The patient is a 58-year-old Malaysian woman who lives in a rural area, where uncontrolled populations of stray and semidomesticated dogs live in close proximity with humans., (© The American Society of Tropical Medicine and Hygiene.)

This study compared the course of infection by Ancylostoma caninum and Ancylostoma braziliense in mixed-breed dogs infected with L3 larvae. Dogs infected with A. caninum eliminated more eggs than did those infected with A. braziliense. A total of 38 % of A. caninum and 44 % of A. braziliense larvae were recovered as adult worms. There were no marked clinical abnormalities in dogs with either infection. A. caninum was associated with anemia and an increased number of circulating neutrophils, whereas infection with A. braziliense led to a decrease in the number of leukocytes. The humoral response against excreted and secreted antigens from adult worms was more sensitive and specific than the response induced with the crude antigen. No immune response was observed for either crude or excreted-secreted (ES) antigens from larvae of either species. A nonspecific response against the crude antigen of A. braziliense was found at 0 and 7 days postinfection and maintained throughout the infection period. However, antibody titers against ES antigens were elevated in A. caninum infection at patency and death, showing that this antigen has a higher specificity. The immune response elicited by infection with A. braziliense in dogs has not been described previously. No significant differences were observed in the infection processes of the two Ancylostoma species, except for the higher number of eggs eliminated from dogs infected with A. caninum, which may indicate a better evolutionary adaptation of the parasite to its host in comparison with A. braziliense.

An experiment was conducted to assess the mucosal response to low-dose superimposed challenge with Ancylostoma ceylanicum. Hamsters were assigned to five treatment groups (1-5 respectively): naïve controls; primary immunizing infection controls; challenge controls; immunized, anthelmintic-treated, challenged group; immunized, superimposed challenge group. Group 4 hamsters were resistant to challenge, whereas most of the challenge inoculum larvae established in Group 5. Villus height and crypt depth measurements were initially markedly divergent between these two groups but over time post-challenge (pc) values for both parameters drew nearer and by day 31 pc they were indistinguishable. The greatest change was experienced by Group 4 which showed increasing inflammation and gut pathology during the challenge infection. Mitotic activity in crypts and mast cell counts in the mucosa were highest in Group 5 on day 10 pc, but there was little to distinguish between Groups 4 and 5 by day 31 pc. Goblet cell, eosinophil and Paneth cell counts were very similar throughout in both groups but, in the case of Paneth cells, they were consistent with a possible role in protective immunity to challenge. Some adult worms survived throughout the period of intense inflammation, emphasizing their tremendous resilience and resistance to mucosal host protective responses.

Hookworm infection is associated with anaemia and malnutrition in many resource-limited countries. Ancylostoma hookworms have previously been shown to modulate host cellular immune responses through multiple mechanisms, including reduced mitogen-mediated lymphocyte proliferation, impaired antigen presentation/processing, and relative reductions in CD4(+) T cells in the spleen and mesenteric lymph nodes. Syrian hamsters were depleted of CD4(+) for up to 9 days following intraperitoneal injection (200 microg) of a murine anti-mouse CD4 monoclonal IgG (clone GK1.5). CD4(+) T-cell-depleted hamsters infected with the hookworm Ancylostoma ceylanicum exhibited a threefold higher mean intestinal worm burden and more severe anaemia than animals that received isotype control IgG. In addition, depletion of CD4(+) T cells was associated with impaired cellular and humoral (serum and mucosal) immune responses to hookworm antigens. These data demonstrate an effector role for CD4(+) T cells in hookworm immunity and disease pathogenesis. Ultimately, these studies may yield important insights into the relationship between intestinal nematode infections and diseases that are associated with CD4(+) T-cell depletion, including HIV.

We assessed the mucosal response of previously infected hamsters to low-dose challenge with the hookworm, Ancylostoma ceylanicum. Hamsters were assigned to five treatment groups (Groups 1-5, respectively): naïve, controls; uninterrupted primary infection from day 0; infected, but treated with anthelmintic on day 35 p.i.; challenge control group given only the second infection on day 63; infected initially, cleared of worms and then challenged. Animals were culled on days 73 and 94 (10 and 31 days after challenge), but additional animals were culled from Group 5 on days 80 and 87. The results showed that villus height declined markedly and progressively over time after challenge in Group 5, whilst depth of the Crypts of Lieberkühn and number of mitotic figures in the crypts increased. Mucosal mast cell numbers were only marginally higher than those in naïve controls and not as high as those in mice with uninterrupted infections. Goblet cell counts showed a major increase, as did eosinophils in relation to naïve controls. Paneth cells were also elevated, but did not change over the course of the experiment. The results also drew attention to the tremendous resilience of hookworms, some adult worms surviving throughout, despite highly inflamed intestines.

Although hookworms are known to stimulate inflammatory responses in the intestinal mucosa of their hosts, there is little quantitative data on this aspect of infection. Here we report the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum. Infection resulted in a marked increase in goblet cells in the intestinal mucosa, which was dependent on the number of adult worms present and was sustained as long as worms persisted (over 63 days) but returned to baseline levels within 7 days of the removal of worms by treatment with ivermectin. Increased mast cell responses were also recorded. Levels were again dependent on the intensity of worm burdens and lasted as long as 63 days after infection. When worms were eliminated, mast cell numbers took over 2 weeks to return to normal. Paneth cell numbers fell soon after infection, the degree of reduction being dependent on the worm burden. After clearance of worms, Paneth cell numbers returned to normal within a week, but then rebounded and numbers rose to higher levels than those in control naïve animals. The time course of the response was similar whether animals experienced a chronic low-intensity infection without loss of worms or a higher intensity infection during the course of which worm burdens were gradually reduced. Clearly, A. ceylanicum was able to induce a marked inflammatory response in its host's intestine which was sustained for over 9 weeks after infection, and which hamsters appeared able to tolerate well. Our data draw attention to the resilience of hookworms which, unlike many other nematodes, are able to survive for many weeks in a highly inflamed intestinal tract.

Changes induced in the circulating and intestinal eosinophil levels and worm load were studied in female Swiss albino mice infected with multiple doses of Ancylostoma caninum larvae. There was a significant rise of both circulating and intestinal eosinophils in all the infected animals when compared with controls. In all the experimental groups of animals, peak eosinophilia was observed during the third week of post-infection and sustained upto 30th day with slight alterations. These studies indicated that eosinophilia will be induced due to anaphylactic reactions in the gut and eosinophilia in multiple infections is an anamnestic response.

Anti-hookworm antibody serologic responses were measured in residents of an Anhui provincial Chinese village where Ancylostoma duodenale is the predominant hookworm. Antibody responses were measured against either soluble infective third-stage larval (L3) or adult antigens. Immunoglobulins of the IgG class, especially IgG4 correlated with both the prevalence and intensity of A. duodenale hookworm infections. In contrast, there was an inverse correlation with IgM, but no correlation with IgA or IgE. Circulating IgG4 antibody responses might serve as a surrogate marker for active A. duodenale hookworm infection.

Ancylostoma duodenale is still the dominant hookworm species in the Mediterranean area, India, China and Japan. In the present study, biopsied materials were taken from the small intestine of 30 patients infected only with A. duodenale and 12 cross matched controls. The results showed some pathological changes in severely infected cases. However, normal or insignificant changes were seen in the enzymatic activity of the intestinal mucosa.

Objective: To elucidate the mechanisms of protective immunity in mice elicited by living hookworm (Ancylostoma caninum third-stage infective larvae (L3)., Methods: The number of migrating infective larvae recovered from the lungs was used as an endpoint for vaccine immunity. The timing of maximal L3 lung entry was determined by counting the number of lung larvae at several time points after infection with 500 or 1000 L3. Mice were immunized either orally or subcutaneously with 500 L3, followed by two boosts of L3 once every two weeks. The immunized mice were challenged orally with 500 L3 one week after the final boost. To evaluate the protective immunity, the number of L3 recovered from the lungs of the immunized mice during the time of maximal larval entry was compared with that of controls. Host immunity was also evaluated by comparing circulating anti-L3 antibodies between immunized and controlled mice, using both enzyme immunoassays and immunoblotting techniques, and by lung histopathology., Results: The peak time of larval entry into the lungs occurred 48 hours after infection. Mice immunized either orally or subcutaneously with L3 exhibited a marked reduction (90.2% and 86.2% respectively) in the number of recovered lung larvae in comparison to controls (P < 0.01). The protection might be associated with circulating anti-L3 antibodies, including antibodies directed against 132-200 kDa L3 antigens, as well as three major antigens ranging from 28 to 51 kDa. Larvae migrating through the lungs of vaccinated mice showed cuticular damages accompanied with host-inflammatory cell invasion., Conclusions: Immunization with living L3 protects mice against lung invasion after challenged with hookworm infection. Vaccine immunity is associated with circulating antibodies against L3 antigens and lung inflammatory responses. The mouse model is potentially useful for developing a hookworm vaccine.

Recent studies in northeastern Australia indicate that enteric infection with Ancylostoma caninum is a leading cause of human eosinophilic enteritis. Much more frequent accompaniments of this infection are obscure abdominal pain with or without blood eosinophilia, while a large part of the population is probably infected asymptomatically. These conclusions are based on extensive serological investigations in patients and control subjects, as well as 15 cases in which single, adult hookworms were identified in situ in patients. In no case has more than one worm been identified, and none has been fully mature, so the infections have never been patent. Aphthous ulcers of the terminal ileum, caecum and colon have been seen in association with this infection and have also been observed in almost 5% of patients who are colonoscoped in north Queensland. Serodiagnosis has relied on an IgG and IgE ELISA using excretory-secretory antigens from adult A. caninum, but Western blot using these antigens to identify IgG4 antibodies to a protein of molecular weight 68 kDa (Ac68) promises to be more specific and sensitive. However, identical antigens appear to be secreted by the anthropophilic hookworms as well. The clinical, public health and biological significance of these findings are discussed in detail.

Ileal ulceration is diagnosed commonly at colonoscopy in northeastern Australia. This observation has occurred contemporaneously with the description of a painful enteritis caused through supposedly rare infections by Ancylostoma caninum, the cosmopolitan dog hookworm. In this study, we describe the morphology and prevalence of ileal ulcers and investigate their cause. There were 182 patients with a distinctive aphthous ileitis highlighted by haemorrhage into the tips of surrounding villi. The annual rate reached 4.7% of ileal examinations. Eosinophilic enteritis was the characteristic, but inconsistent, histological appearance. Ulceration was linked to infection by A. caninum through serological testing and, in three patients, by the retrieval of single hookworms. Affected patients were also more likely to own a dog (53%) than the total colonoscopic population (43%; P < 0.02). The monthly rates of diagnosis were related to climate with fewest recordings during winter. Pain was the chief indication for colonoscopy (49%), proportionally higher than in controls (28%; P < 0.0001). Patients with ulcers had increased blood eosinophil, immunoglobulin E and serological values. These results were higher still in the subset with pain compared with the rest. Most patients, however, had colonoscopy performed for unrelated reasons, such as bowel cancer surveillance, and were identified as having subclinical ulceration (rate 3.2%). The findings establish a distinctive aphthous ileitis characterized by tissue eosinophilia. The cause has been tentatively linked to canine hookworms. It is proposed that infection in this geographic region is common and only rarely does a painful enteritis develop as a consequence of hypersensitization.

It retards growth and intellectual development in millions of children yet is largely ignored by researchers. New findings suggest excellent possibilities for a vaccine.

Using Watson's intestinal capsule small intestinal mucosal biopsies were taken from 30 patients with parasitologically proved pure ancylostomiasis and 12 normal controls. Biopsies were examined pathologically and histochemically for the enzymes, Alkaline phosphatase, succinic dehydrogenase, non specific esterases and acid phosphatase. The study revealed normal histological as well as normal enzymatic activity of the intestinal mucosa.

During the period January to April 1977, 34 dogs were autopsied and examined for hookworms in Yogyakarta, Indonesia, A. caninum was found in all 34 dogs (100%), A. ceylanicum in 33 dogs (97.1%) and A. braziliense in 7 dogs (20.6%). The dogs yielded 2,213 ancylostomes, of which 1,188 were A. caninum, 989 A. ceylanicum and only 36 were A. braziliense. All species of hookworms appeared to be more concentrated in the jejunum than in the duodenum or ileum. The lesion produced, proved to be a traumatic injury and was mostly superficial and never reached the submucosa.

The alterations of mast cell counts induced in the gut of swiss albino mice by infection with Ancylostoma caninum larvae have been studied, both in single and multiple infections. It was found that mast cell rise was greater in females than males. In singly infected animals, mast cell increase was influenced by the infective dose of inoculated larvae. In case of repeatedly infected mice, mast cell counts were decreased because of their earlier degranulation.

A variety of cell types in the intestinal mucosa of mice has been observed following oral infection with Ancylostoma ceylanicum and the changes in their numbers quantified. The number of lamina propria mast cells increased following infection and reached a peak at 4 days; by 21 days the numbers had returned to normal levels. Globule leucocytes displayed a similar marked response but the numbers returned to within normal limits 7 days after infection. Granular intraepithelial lymphocytes did not alter significantly during the period of observation whereas non-granular intraepithelial lymphocytes declined in numbers following infection and were significantly decreased by 3 weeks. Goblets cells showed a similar response to infection as lamina propria mast cells and reached a peak level at 4 days. Following initial fluctuations in eosinophil numbers there was a substantial increase in the numbers of these cells with peak levels being reached at 21 days; eosinophil numbers then remained elevated for the duration of observation. Partial villous atrophy was seen 4 days after the arrival of larvae in the small intestine with maximum reduction in the length of villi at 7 days. Thereafter, there was a return to normal length followed by a significant elongation in villus length 28 days after infection. The influences of these various cell types on the expulsion of intestinal parasites are discussed.

Ancylostoma ceylanicum infection in golden hamsters (Mesocricetus auratus) caused marked biochemical and histopathological derangements. Jejunum, the primary site of infection, showed pronounced alterations compared with liver. Though the biochemical composition of jejunum was not significantly altered, activities of a few lysosomal enzymes were enhanced during hookworm infection. Marked damage to mitochondrial and microsomal membranes was reflected in changes in the activities of the marker enzymes from jejunal tissue. Lipid content, especially phospholipids and neutral lipids of hepatic tissue, exhibited marked elevation. Levels of hexokinase, phosphofructokinase, and lactate dehydrogenase were enhanced in jejunal as well as hepatic tissues, indicating activation of the glycolytic machinery during hookworm infection. A decrease in the levels of mucosal disaccharidases indicated damage to intestinal brush border membranes. However, alkaline phosphatase activity was increased in intestinal mucosa during the infection. Light microscopic examination of jejunal tissue revealed peeling off of the upper epithelial layer, activation of the goblet cells, and thickening of muscularis mucosa. However, hepatic tissue did not show gross alterations, except for slight necrosis in the centrilobular region.

Remarkable hypertrophic changes were observed in the adrenal and thyroid glands of male mice after 7 to 16 days of infection with the filariform larvae of Ancylostoma caninum, but not in females. The adrenal gland revealed enormous enlargement of cells in the zonae glomerulosa, fasciculata and medulla, and also the formation of big vacuoles in the glomerular zone, whereas the thyroid gland showed distinctly hypertrophied follicular epithelium with marked acolloidal condition and follicular mass formation. These changes in the cellular diameter were highly significant (P greater than 0.001 to less than 0.005). The possible reasons for hyperactivity of these glands are discussed.