Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia.

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5-10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.

Morphological differentiation of eggs of Ancylostoma caninum, Ancylostoma tubaeforme, and Ancylostoma braziliense from dogs and cats in the United States.

The establishment of cat- and dog-derived laboratory strains of Ancylostoma braziliense allowed for a morphological comparison of the eggs of A. braziliense, Ancylostoma caninum, and Ancylostoma tubaeforme. The length, width, and perimeter were determined for images of 10 eggs each of A. braziliense from the feces of a dog infected with a canine isolate and a cat infected with a feline isolate, A. caninum from dog feces, and A. tubaeforme from cat feces. The specific identity of the eggs was verified by polymerase chain reaction and restriction fragment length polymorphism by using HinfI and RsaI restriction digests followed by gel electrophoresis and sequencing. The mean (±SD) length, width, and perimeter and the length-to-width ratio (±SD) (all measurements are in micrometers) for the eggs of each species were as follows: A. braziliense eggs (combined cat and dog source), 53.03 ± 2.33, 36.37 ± 1.35, 140.43 ± 2.56, and 1.46 ± 0.11; A. caninum eggs, 63.92 ± 5.28, 39.21 ± 1.52, 161.99 ± 9.30, and 1.63 ± 0.13; and A. tubaeforme eggs, 61.44 ± 3.05, 39.14 ± 1.40, 157.98 ± 5.81, and 1.57 ± 0.08. The eggs of A. braziliense were significantly (P < 0.001) smaller than the eggs of A. caninum and A. tubaeforme in all dimensions. Thus, the eggs seem to be readily distinguishable using light microscopy, thereby aiding in species identification in fecal samples for a more comprehensive clinical picture and assessment of zoonotic risk.

Scanning electron microscopy of Ancylostoma spp. dog infective larvae captured and destroyed by the nematophagous fungus Duddingtonia flagrans.

The interaction between the nematode-trapping fungus Duddingtonia flagrans (isolate CG768) against Ancylostoma spp. dog infective larvae (L(3)) was evaluated by means of scanning electron microscopy. Adhesive network trap formation was observed 6h after the beginning of the interaction, and the capture of Ancylostoma spp. L(3) was observed 8h after the inoculation these larvae on the cellulose membranes colonized by the fungus. Scanning electron micrographs were taken at 0, 12, 24, 36 and 48 h, where 0 is the time when Ancylostoma spp. L(3) was first captured by the fungus. Details of the capture structure formed by the fungus were described. Nematophagous Fungus Helper Bacteria (NHB) were found at interactions points between the D. flagrans and Ancylostoma spp. L(3). The cuticle penetration by the differentiated fungal hyphae with the exit of nematode internal contents was observed 36 h after the capture. Ancylostoma spp. L(3) were completely destroyed after 48 h of interaction with the fungus. The scanning electron microscopy technique was efficient on the study of this interaction, showing that the nematode-trapping fungus D. flagrans (isolate CG768) is a potential exterminator of Ancylostoma spp. L(3).

Predation of Ancylostoma spp. dog infective larvae by nematophagous fungi in different conidial concentrations.

In the present work, it was evaluated the in vitro effect of 12 isolates from the fungal species Arthrobotrys, Duddingtonia, Nematoctonus and Monacrosporium genera in different conidial concentrations on the capture of Ancylostoma spp. dog infective larvae (L(3)), on 2% water-agar medium at 25 degrees C, at the end of a period of 7 days. The concentrations used for each nematophagous fungus were 1000, 5000, 10,000, 15,000 and 20,000conidia/Petri dish plated with 1000 Ancylostoma spp. L(3). All nematode-trapping fungi isolates tested reduced the averages of the uncaptured Ancylostoma spp. L(3) recovered, with the increase of the fungal inoculum concentration, in comparison to the fungus-free control (p<0.05). The adhesive network producing species were better predators than the constricting ring or adhesive knob producing species. Duddingtonia flagrans (Isolate CG768) was the most effective, reducing the averages of the uncaptured Ancylostoma spp. L(3) recovered in 92.8%, 96.3%, 97.5%, 98.3% and 98.9%, respectively in five fungal inoculum concentrations established. Other effective nematophagous fungi were Arthrobotrys robusta (Isolate I31), which reduced the averages of the uncaptured Ancylostoma spp. L(3) recovered in 85.4%, 88.3%, 90.7%, 92.5% and 95.2%, and Arthrobotrys oligospora (Isolate A183), with reductions of 66.6%, 79.8%, 86.8%, 89.5% and 90.8%, respectively for both, in the five fungal inoculum concentrations established. No difference was found between Isolates A183 and I31 in the conidial concentrations of 15,000/Petri dish. Nematoctonus robustus (Isolate D1) and Arthrobotrys bronchophaga (Isolate AB) had the smallest percentages of reduction among the tested isolates and showed the lowest predacious activity. The Isolates CG768, I31 and A183 were considered potential biological control agents of Ancylostoma spp. dog free-living stages, being directly influenced by the fungal inoculum concentration.

Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes.

Mitochondrial genome sequences provide useful markers for investigating population genetic structures because of their maternal inheritance and high evolutionary rates. There is, however, a paucity of information on mitochondrial genomes for many parasitic organisms, including nematodes, which appears to relate mainly to technical limitations and (for modestly funded laboratories) the cost associated with full mitochondrial genome sequencing. In this article, we describe a simple, relatively inexpensive long-PCR approach for the amplification (using two sets of primers) of the entire mitochondrial genome from individual parasitic nematodes for subsequent sequencing, which overcomes these limitations. We employed two species of human hookworm (Ancylostoma duodenale and Necator americanus; order Strongylida) to establish the long-PCR conditions, and then extended its use to a number of other species of parasitic nematode of the class Secernentea (orders Strongylida, Ascaridida and Rhabditida). The long-PCR method for the amplification of the entire mitochondrial genome from single nematodes, coupled with direct sequencing of amplicons, provides a useful tool for the comparative analysis of genome organisation and evolution of a range of nematode groups. It also creates a platform for molecular ecological and population genetic studies.

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae.

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L3). Mice were vaccinated by oral immunization with 500 L3 (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L3) 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L3 from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L3 within 2 h, and over 55% of the L3 were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24-72 h after challenge, almost all of the L3 recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L3. In contrast, the L3 recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L3-vaccinated mice.

Giardia trophozoites in dog hookworm, Ancylostoma caninum: accident or hyperparasitism?

During the course of transmission electron microscopic studies of adult Ancylostoma caninum removed from a dog, several Giardia trophozoites were found in sections of the buccal cavity, oesophagus and intestine of several hookworms. Although the protozoa appeared viable, this unusual finding probably represents accidental uptake by, rather than an established infection of, the hookworm. It is feasible, however, that the trophozoites might have survived and even multiplied in this aberrant site.

Electron and light microscopy of peritoneal cellular immune responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae.

The role of peritoneal macrophages in a murine model of immunity to living hookworm third-stage larvae (L3) was investigated. Mice immunized orally with 500 L3 once every 2 weeks for three times were challenged intraperitoneally with 2000 L3 1 week after the final immunization. The challenged larvae were collected from the peritoneal cavity at intervals between 2 and 72 h and then examined by inverted light microscopy, scanning electron microscopy and transmission electron microscopy. Peritoneal cellular responses in non-immunized mice served as negative controls. The numbers of peritoneal macrophages in immunized mice were 6-7-fold higher than in non-immunized mice. In the peritoneal cavity of immunized mice, host macrophages adhered to the challenged L3 within 2 h and created a cocoon-like encasing which surrounded the parasite. Extensive damage to the L3 was observed which included swelling, collapse and deformation of the larval cuticle. Lysis and vacuolization of the parasite's internal structures were also observed. In contrast, no significant cellular adherence and damage were observed in L3 recovered from non-immunized mice. L3-specific antibody levels were also elevated in the peritoneum of immunized mice relative to non-immunized controls. These studies implicate macrophages as important effector cells in hookworm larval vaccine immunity.

A distinctive aphthous ileitis linked to Ancylostoma caninum.

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.

Ancylostoma genettae, A. protelesis, A. somaliense: three new species from wild Carnivora in the Somali Republic.

Ancylostoma braziliense was found in Somalia in Acinonyx jubatus, Canis familiaris, C. mesomelas, Crocuta crocuta, Felis catus, F. libyca, Genetta genetta, Otocyon megalotis, Proteles cristatus; A. caninum in A. jubatus, C. familiaris, C. mesomelas, C. crocuta; A. duodenale in C. crocuta; A. iperodontatum in Lynx caracal; A. paraduodenale in Felis serval; A. tubaeforme in A. jubatus, F. catus, F. libyca; Arthrocephalus gambiense in Ichneumia albicauda; Uncinaria parvibursata in Mellivora capensis. In addition, three new species of Ancylostoma were collected: A. genettae in Genetta genetta, A. protelesis in Proteles cristatus, A. somaliense in Canis mesomelas. These new species are described and illustrated.

[Scanning electron microscopic observations on the copulatory spicules of the male Necator americanus and Ancylostoma duodenale].

Scanning electron microscopic observations were made on the morphological structures of the two copulatory spicules of the male Necator americanus and Ancylostoma duodenale. In both species, one of the two copulatory spicules was in the shape of a concave groove and the other, in the shape of an oblate tube. Owing to the difference in the concavity of the groove shaped copulatory spicule between the two species as shown by the cross sections, Necator americanus usually appear to have only one copulatory spicule whereas Ancylostoma duodenale usually show two separate copulatory spicules in appearance (Figs 1-10).

Ultrastructure of the adults of Bunostomum phlebotomum (Nematoda: Ancylostomatidae)

The morphology of the male and female of Bunostomum phlebotomum are described based on scanning electron microscope (SEM) observations. The attachment of the worms to the small intestinal mucosa and during the copula were observed. Structures of the bucal capsule and genital organs were also studied

Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults.

A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent "sphaerocrystals" located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

Axenic growth in vitro of Ancylostoma duodenale to the filariform (resting) larvae.

Large numbers of free-living stages of Ancylostoma duodenale can be grown in vitro on formalin-killed Escherichia coli supplemented with cholesterol and Dulbecco's minimum essential medium (DMEN). Eggs at 28 degrees C hatched and released first-stage larvae in 24 hr. First-stage larvae molted in 36 hr. at 28 degrees C. Second-stage larvae, molted in 2-3 days at 28 degrees C, but retained the old cuticle. Third-stage larvae characterized by the presence of a buccal cap capsule; sheath; filariform esophagus and absence of lips. Lateral alae were observed in all free-living stages.

Human eosinophilic enteritis caused by dog hookworm Ancylostoma caninum.

An epidemic of eosinophilic enteritis (93 cases) has occurred in Townsville, northern Queensland, Australia. A hookworm was found attached to a resected, inflamed ileal segment from 1 patient but the species could not be identified. An adult hookworm of species Ancylostoma caninum was recovered at colonoscopy from the terminal ileum of a later patient. All of 38 patients interviewed in an epidemiological survey described behaviour which could have exposed them to infective larvae of this widespread dog parasite.

Scanning electron microscopy of Ancylostoma ceylanicum and its site of attachment to the small intestinal mucosa of the dog.

The relationship between the hookworm Ancylostoma ceylanicum and the host intestinal mucosa in situ has been examined by using an in vivo fixation technique and scanning electron microscopy. Heads of the adult worms were buried deeply in the mucosa of the dog's small bowel. At some sites of attachment there was severe atrophy and ulceration of the immediately surrounding villi. Superficial deposits of fibrillar material with many enmeshed erythrocytes were associated with these attachment sites. When adult worms became removed after fixation, large numbers of erythrocytes could be seen at the base of the ulcers. Examination of the morphological details of adult A. ceylanicum revealed the characteristic mouthparts of large outer ventral teeth and smaller inner teeth. The distance between adjacent transverse cuticular striations was measured as 5.2 microns. The wide separation of the externolateral ray from the closely related mediolateral and posterolateral rays was illustrated together with the functi characteristic mouthparts of large outer ventral teeth and smaller inner teeth. The distance between adjacent transverse cuticular striations was measured as 5.2 microns. The wide separation of the externolateral ray from the closely related mediolateral and posterolateral rays was illustrated together with the functi characteristic mouthparts of large outer ventral teeth and smaller inner teeth. The distance between adjacent transverse cuticular striations was measured as 5.2 microns. The wide separation of the externolateral ray from the closely related mediolateral and posterolateral rays was illustrated together with the functional relationship of male and female copulating worms.

Transmission electron microscopical studies of the site of attachment of Ancylostoma ceylanicum to the small bowel mucosa of the dog.

The host-parasite interaction at the site of attachment of the adult hookworm, Ancylostoma ceylanicum, to the small bowel mucosa of the dog has been examined by transmission electron microscopy. The lamina propria around the heads of the worms showed intense infiltration with neutrophilic and eosinophilic leucocytes, and plasma cells were also prominent. Erythrocytes were extravasated within the lamina propria while the portions of mucosa engulfed into the buccal cavities of the worms were necrotic and included aggregates of collagen fibres. Within the lumina of the worms were erythrocytes in varying stages of lysis together with other necrotic debris. The mucous membrane adjacent to the heads of the worms was ulcerated while more distant enterocytes were small, distorted and displayed various degrees of microvillar loss. Many mucosal blood vessels had platelet aggregates within their lumina but no fibrin deposition was observed. Vessels were often seen in the bases of the ulcers and were prone to rupture. Erythrocytes and leucocytes were seen in the dog intestinal lumen in close proximity to the bodies of the worms.

Endotube-brush border complexes dissected from the intestines of Haemonchus contortus and Ancylostoma caninum.

A procedure for the blunt dissection of the syncytial intestine of Haemonchus contortus to separate the endotube-brush border complex is described. A similar complex can be obtained from the partially cellular intestine of Ancylostoma caninum. The structural features of the complexes from the two sources are compared. The main differences are in the retention of terminal bars by the endotube from A. caninum and in the extent and organization of the microfilaments associated with the basal side of the endotubes.