A New Species of Ancylostoma (Nemata: Strongylida: Ancylostomatidae) from Two Species of Ctenomys in Lowland Bolivia.

From the small intestines of both Ctenomys boliviensis and Ctenomys steinbachi collected from August 1984 through June 1990 from the eastern lowlands of the Department of Santa Cruz, Bolivia a total of 36 specimens of Ancylostoma were recovered. Morphological investigation and comparisons with known species described and reported from mammals in the Neotropical Region show that this is an undescribed species, herein described as new. These nematans were collected from individuals of C. steinbachi collected from near a locality called Caranda (northwest of Santa Cruz de la Sierra) and from C. boliviensis from near Santa Rosa de la Roca (northeast of Santa Cruz de la Sierra) and from cajuchis collected from 3 km west of Estación El Pailón, 30 km east of Santa Cruz de la Sierra. The new species of Ancylostoma differs from all other species of Ancylostoma known from the Neotropical Region in the presence of paired sub-terminal papillae on the dorsal ray of males.

[Differentiating the morphology of infective larvae between two human hookworms].

The morphological differentiation of the infective larvae between human Ancylostoma duodenale and Necator americanus is of great significance for the epidemiological survey of hookworm diseases and human parasitology teaching. Understanding of features of the oral spear and transverse lines on the tunica vaginalis is able to accurately differentiate the infective larvae between these two human hookworms.

Ancylostoma ailuropodae n. sp. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China.

BACKGROUND: Hookworms belonging to the genus Ancylostoma (Dubini, 1843) cause ancylostomiasis, a disease of considerable concern in humans and domestic and wild animals. Molecular and epidemiological data support evidence for the zoonotic potential among species of Ancylostoma where transmission to humans is facilitated by rapid urbanization and increased human-wildlife interactions. It is important to assess and describe these potential zoonotic parasite species in wildlife, especially in hosts that have physiological similarities to humans and share their habitat. Moreover, defining species diversity within parasite groups that can circulate among free-ranging host species and humans also provides a pathway to understanding the distribution of infection and disease. In this study, we describe a previously unrecognized species of hookworm in the genus Ancylostoma in the giant panda, including criteria for morphological and molecular characterization. METHODS: The hookworm specimens were obtained from a wild giant panda that died in the Fengtongzai Natural Reserve in Sichuan Province of China in November 2013. They were microscopically examined and then genetically analyzed by sequencing the nuclear internal transcribed spacer (ITS, ITS1-5.8S-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes in two representative specimens (one female and one male, FTZ1 and FTZ2, respectively). RESULTS: Ancylostoma ailuropodae n. sp. is proposed for these hookworms. Morphologically the hookworm specimens differ from other congeneric species primarily based on the structure of the buccal capsule in males and females, characterized by 2 pairs of ventrolateral and 2 pairs of dorsolateral teeth; males differ in the structure and shape of the copulatory bursa, where the dorsal ray possesses 2 digitations. Pairwise nuclear and mitochondrial DNA comparisons, genetic distance analysis, and phylogenetic data strongly indicate that A. ailuropodae from giant pandas is a separate species which shared a most recent common ancestor with A. ceylanicum Looss, 1911 in the genus Ancylostoma (family Ancylostomatidae). CONCLUSION: Ancylostoma ailuropodae n. sp. is the fourth species of hookworm described from the Ursidae and the fifteenth species assigned to the genus Ancylostoma. A sister-species association with A. ceylanicum and phylogenetic distinctiveness from the monophyletic Uncinaria Frölich, 1789 among ursids and other carnivorans indicate a history of host colonization in the evolutionary radiation among ancylostomatid hookworms. Further, phylogenetic relationships among bears and a history of ecological and geographical isolation for giant pandas may be consistent with two independent events of host colonization in the diversification of Ancylostoma among ursid hosts. A history for host colonization within this assemblage and the relationship for A. ailuropodae n. sp. demonstrate the potential of this species as a zoonotic parasite and as a possible threat to human health. The cumulative morphological, molecular and phylogenetic data presented for A. ailuropodae n. sp. provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms.

The crowding effect in Ancylostoma ceylanicum: density-dependent effects on an experimental model of infection.

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.

Obtaining an isolate of Ancylostoma braziliense from dogs without the need for necropsy.

Isolation of a specific Ancylostoma species typically requires death of the source animal, or holding an animal long enough to collect feces after treatment, for worm recovery and identification. The reason for collecting worms is that the eggs are not easy to distinguish morphologically. In keeping with the 3 Rs of laboratory animal research (reduction, refinement, replacement), the objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time field-collected samples of canine feces without the need for killing the host. During a collection trip to Florida, fecal samples (n  =  148) were collected and identified as containing eggs of Ancylostoma species (n  =  64) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 2 samples were identified as A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Larval cultures were initiated from these samples, and larvae from the cultures were returned to New York and used to inoculate a purpose-bred kitten with the goal of inhibiting the growth of any contaminating Ancylostoma caninum that might be present in the culture. The infection was patent at 15 days, and eggs were identified as A. braziliense by RFLP and DNA sequencing. Using forceps during endoscopy, 2 adult worms (1 male, 1 female) were recovered from the cat and identified morphologically as A. braziliense . Larvae were cultured from the feces of this cat and used to infect a laboratory-reared beagle dog. Additionally, worms recovered from the feces of the cat post-treatment were confirmed to be A. braziliense , except for 1 female A. caninum containing infertile eggs. The dog (patent 14 days post-infection) was also infected with A. braziliense as determined by RFLP and DNA sequencing of eggs and cultured larvae. Both the cat and dog were treated, verified to be no longer shedding eggs, and then placed into adoptive homes.

Identification and characterization of a serine protease inhibitor with two trypsin inhibitor-like domains from the human hookworm Ancylostoma duodenale.

Protease inhibitors play important roles in the parasitic nematodes' survival within their host, in the development and reproduction of the parasites. The present study described the isolation, identification, and characterization of a novel member of the Ascaris family of serine protease inhibitors, designated AduTIL-1, from the human hookworm Ancylostoma duodenale. AduTIL-1 is composed of a signal sequence and two trypsin inhibitor-like (TIL) domains, which showed the highest similarity with OdmCRP, a putative serine protease inhibitor with two TIL domains in Oesophagostomum dentatum. Each TIL domain of the AduTIL-1 was expressed in Escherichia coli, and their inhibitory activities against serine proteases from animals and human were characterized, respectively. Both of the two TIL domains inhibited human neutrophil elastase and pancreatic trypsin, but different in effectiveness. Although the first TIL domain of AduTIL-1 inhibited bovine pancreatic chymotrypsin (Ki=18.0 nM), both of the two domains showed no inhibitory activity against the human pancreatic chymotrypsin. Immunohistochemical studies demonstrated that AduTIL-1 was localized in esophagus, intestine, and cuticular surface of the adult worms. These results suggested that AduTIL-1 may be involved in the survival of A. duodenale in host by targeting related digestive enzymes and neutrophil elastase.

A case of mistaken identity--reappraisal of the species of canid and felid hookworms (Ancylostoma) present in Australia and India.

This study serves to clarify the current status of canid and felid Ancylostoma species present in Australia. The morphological identification of A. ceylanicum from cats for the first time in Townsville, Australia, appears to be in error, together with the genetic markers provided for the species. Morphological and genetic data presented herein provide strong evidence that the hookworms from cats in Towsville are not A. ceylanicum as previously identified (i.e. the first report of this species in Australia), but are A. braziliense. Therefore the subsequent genetic markers established for A. ceylanicum in subsequent molecular studies based on these Townsville specimens should also be attributed to A. braziliense. Based on this information, a study of canine hookworm species present in northern India is also in error and it is apparent that the hookworms found in this region are those of A. ceylanicum. The distribution of A. braziliense and A. ceylanicum in the Americas and Asia Pacific region is discussed together with the importance of combining parasite morphology with genetic data for parasite diagnosis in epidemiological studies.

Short report: an agar plate method for culturing hookworm larvae: analysis of growth kinetics and infectivity compared with standard coproculture techniques.

An agar plate (AP) method has been developed for culturing infectious larvae of the hookworm Ancylostoma ceylanicum. The third-stage larvae reared using the AP method displayed similar morphology to those cultured using Baermann or Harada-Mori coproculture techniques. The yield of viable larvae from the AP method (50%) was comparable to that of the Baermann (47%), and both were superior to Harada-Mori (2.1%). Third-stage larvae cultured by the AP method established patent infection in naturally permissive laboratory hosts, although the yield of adult worms was reduced compared with animals infected with L3 obtained by Baermann culture. The AP method is useful for defining growth requirements for hookworm development, as well as characterizing the effects of bacterially expressed compounds on hookworm larvae in vivo.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

A multi-enzyme cascade of hemoglobin proteolysis in the intestine of blood-feeding hookworms.

Blood-feeding pathogens digest hemoglobin (Hb) as a source of nutrition, but little is known about this process in multicellular parasites. The intestinal brush border membrane of the canine hookworm, Ancylostoma caninum, contains aspartic proteases (APR-1), cysteine proteases (CP-2), and metalloproteases (MEP-1), the first of which is known to digest Hb. We now show that Hb is degraded by a multi-enzyme, synergistic cascade of proteolysis. Recombinant APR-1 and CP-2, but not MEP-1, digested native Hb and denatured globin. MEP-1, however, did cleave globin fragments that had undergone prior digestion by APR-1 and CP-2. Proteolytic cleavage sites within the Hb alpha and beta chains were determined for the three enzymes, identifying a total of 131 cleavage sites. By scanning synthetic combinatorial peptide libraries with each enzyme, we compared the preferred residues cleaved in the libraries with the known cleavage sites within Hb. The semi-ordered pathway of Hb digestion described here is surprisingly similar to that used by Plasmodium to digest Hb and provides a potential mechanism by which these hemoglobinases are efficacious vaccines in animal models of hookworm infection.

Chemo- and thermosensory neurons: structure and function in animal parasitic nematodes.

Nematode parasites of warm-blooded hosts use chemical and thermal signals in host-finding and in the subsequent resumption of development. The free-living nematode Caenorhabditis elegans is a useful model for investigating the chemo- and thermosensory neurons of such parasites, because the functions of its amphidial neurons are well known from laser microbeam ablation studies. The neurons found in the amphidial channel detect aqueous chemoattractants and repellants; the wing cells-flattened amphidial neurons-detect volatile odorants. The finger cells-digitiform amphidial neurons-are the primary thermoreceptors. Two neuron classes, named ADF and ASI, control entry into the environmentally resistant resting and dispersal dauer larval stage, while the paired ASJ neurons control exit from this stage. Skin-penetrating nematode parasites, i.e. the dog hookworm Ancylostoma caninum, and the threadworm, Strongyloides stercoralis, use thermal and chemical signals for host-finding, while the passively ingested sheep stomach worm, Haemonchus contortus, uses environmental signals to position itself for ingestion. Amphidial neurons presumably recognize these signals. In all species, resumption of development, on entering a host, is probably triggered by host signals also perceived by amphidial neurons. In the amphids of the A. caninum infective larva, there are wing- and finger-cell neurons, as well as neurons ending in cilia-like dendritic processes, some of which presumably recognize a sequence of signals that stimulate these larvae to attach to suitable hosts. The functions of these neurons can be postulated, based on the known functions of their homologs in C. elegans. The threadworm, S. stercoralis, has a complex life cycle. After leaving the host, soil-dwelling larvae may develop either to infective larvae (the life-stage equivalent of dauer larvae) or to free-living adults. As with the dauer larva of C. elegans, two neuron classes control this developmental switch. Amphidial neurons control chemotaxis to a skin extract, and a highly modified amphidial neuron, the lamellar cell, appears to be the primary thermoreceptor, in addition to having chemosensory function. The stomach worm, Haemonchus contortus, depends on ingestion by a grazing host. Once ingested, the infective larva is exposed to profound environmental changes in the rumen. These changes stimulate resumption of development in this species. We hypothesize that resumption of development is under the control of the ASJ neuronal pair. Identification of the neurons that control the infective process could provide the basis for entirely new approaches to parasite control involving interference with development at the time and place of initial host-contact.

Apparent feeding behaviour of ensheathed third-stage infective larvae of human hookworms.

The third-stage ensheathed infective larvae (L3) of hookworms are reputed to be non-feeding stages, and the L2-derived sheath is considered to be a barrier to macromolecular uptake and secretion. During a study of the lectin-binding characteristics of en- and exsheathed L3 larvae of Ancylostoma duodenale, fluorescein isothiocyanate conjugated lectins (con A and Ulex europaeus agglutinin) were repeatedly seen in the oesophageal lumen, as well as on the surface of L3 larvae encased in the L2 sheaths. Repetition of these experiments using Necator americanus L3 larvae yielded similar results. These observations suggest that ensheathed hookworm larvae can obtain/utilise and secrete macromolecules, despite the presence of a sheath.

Extrusion of the residual body in spermatids of Ancylostoma caninum (Nematoda, Strongyloidea).

Transmission electron microscopy reveals that the spermatocytes of the hookworm Ancylostoma caninum contain an abundance of Golgi complexes, ribosomes, specialized membranous organelles, and long strands of smooth endoplasmic reticulum. These organelles remain abundant until the early spermatid stage of sperm development, when they reach their maximum abundance and maturity and the production of new ones ceases. Golgi complexes, ribosomes, and excess SER, which are not functional after this stage, become segregated and confined to the posterior portion of the spermatid in a polar lobe. Later, the polar lobe together with excess cytoplasmic matrix is bound by a membrane and dissociated from the spermatid as a residual body. The spermatid is then devoid of Golgi complexes and ribosomes. Formation of residual bodies as sperm cells mature may be considered a type of cell excretion.

Differences in lipid granulation as the basis for a morphologic differentiation between third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum.

Differences in the distribution of lipid granules between unstained third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum cultured at 15 C was found to be an effective means for differentiating these 2 species of canine hookworms. In contrast, larvae cultured at 22 C were less easily differentiated based on the distribution of lipid granules. After culturing at 15 C, third-stage larvae of U. stenocephala were motile and exhibited 32 well-demarcated intestinal cells which contained intracellular lipid granules. Intestinal cells were easily visualized due to the absence of extraintestinal lipid granulation. Ancylostoma caninum third-stage larvae cultured under similar conditions were significantly less motile and contained extraintestinal accumulations of lipid granules which obscured intestinal cells. Both species exhibited an overall decrease in lipid granulation during a 14-day observation period following culture at 15 C. Morphologic differentiation of these 2 species after 14 days was based on the absence of intra- and extra-intestinal lipid in U. stenocephala and the presence of some lipid granules in both these locations in A. caninum. The first- and second-stage larvae of both species cultured at 15 C exhibited dense accumulations of extraintestinal lipid granules and were morphologically indistinguishable. This suggests that the observed difference in lipid granulation between the third-stage larvae of U. stenocephala and A. caninum cultured at 15 C is due to differences in lipid utilization during the third stage rather than differences in lipid synthesis by the first- and second-stage larvae and is related to the adaptation of these parasites to their respective climatic regions.

[Comparative value of egg size and larval morphology in the diagnosis of species of ancylostomids. Existence of abnormal larvae]. / Intérêt comparé de la taille des oeufs et de la morphologie des larves dans le diagnostic des espèces d'ankylostomidés. Existence de larves anormales.

The values of eggs mensuration and results of coproculture have been compared for the identification of species of ancylostomia infestations. Results are confirming the necessity of coproculture for this diagnosis when the size of eggs is not inferior to 61 mu or superior to 72 mu. One case of morphological anomaly of some strongyloïd larvae of Necator americanus has been related.

Scanning electron microscopy of the penetration of newborn mouse skin by Strongyloides ratti and Ancylostoma caninum larvae.

The scanning electron microscopical appearances of infective larvae of Strongyloides ratti and Ancylostoma caninum have been described. Particularly noteworthy was the tail of S. ratti which was found to have a distal aperture surrounded by a row of eight projections. The penetration of larvae through newborn mouse skin was investigated. S. ratti larvae forced its way rapidly and directly through the stratum corneum. No larvae of A. caninum were observed in stages of partial penetration but the occasional empty sheath was seen.

Human Ancylostoma infections in Nigeria.

An estimate of the proportion of infections with Ancylostoma duodenale and Necator americanus by the examination of larvae collected from the stools of 220 persons infected with hookworm was made. Fifty-nine per cent of the infections were due to N. americanus only: A. duodenale always occurred in association with N. americanus. In 12% of the infections it is suggested that there were approximately three female A. duodenale to 95 female N. americanus. The highest numbers of A. duodenale in mixed infections (28 female A. duodenale with 45 female N. americanus) occurred in only 1% of the infections. Evidence is given that egg size can be important in the differentiation of the two species. A method of calculating the proportion of the two hookworms, and hence of other intestinal nematodes, in mixed infections in an individual as well as in the general population, is also described.