Hookworm: developmental biology of the infectious process.

Hookworms cause severe anemia and malnutrition in developing countries of the tropics, with an estimated one billion people infected worldwide. An in vitro system that models the early events of infection has provided new information about the linkage between the infectious process and the parasite's developmental biology. The cloning and expression of Ancylostoma secreted protein, ASP 1 - a secreted molecule associated with these developmental processes - is an example of how this system allows us to dissect the infectious process at the molecular level.

Identification of a putative structural gene for cathepsin D in Caenorhabditis elegans.

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.

Decline in protease activities with age in the nematode Caenorhabditis elegans.

The activities of 3 lysosomal proteases in the nematode Caenorhabditis elegans are markedly lower in older animals. The aspartyl protease cathepsin D declines about 10-fold from day 3 (early adulthood) to day 11 (near the mean lifespan); this reflects a net decline in the amount of cathepsin D antigen. The specific activity of the thiol protease cathepsin Ce1 declines about 2.5-fold over the same period, and the specific activity of the thiol protease cathepsin Ce2 declines about 8-fold. The activity of a new non-lysosomal protease, designated cathepsin CeX, is invariant with age. The data are consistent with the hypothesis that reduced protease activity in older animals may cause a decline in the rate of protein turnover with age, but do not prove this hypothesis.

Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum.

Invading infective third-stage larvae (L3) of parasitic nematodes execute a series of programmed developmental events in response to a host-specific signal encountered during infection. One of these early events is the release of excretory/secretory products. Using an in vitro feeding assay that mimics these early events of infection, a protein released by in vitro activated larvae of the hookworm Ancylostoma caninum was identified. This protein, Ac-ASP-2, was partially sequenced, and the cDNA encoding it isolated by PCR and screening of an A. caninum L3 cDNA library. The Ac-asp-2 cDNA encodes a protein of 219 amino acids that is related to a previously identified protein, Ac-ASP-1, from hookworms. Both molecules are members of an evolutionarily diverse family of molecules that include the venom allergens of the Hymenoptera, and the testes specific proteins/sperm-coating glycoproteins of mammals. Homologues are present in nearly all nematodes tested, as demonstrated by PCR-hybridization and database searching. The Ac-asp-2 mRNA is synthesized in all life history stages, but the gene product is released only by L3 activated to feed in vitro. The wide distribution of the Ac-asp-2 in nematodes and its release in response to host specific signals suggests that Ac-ASP-2 serves an important function in nematode physiology and development, and possibly in the infective process of parasitic species.

Ancylostoma caninum anticoagulant peptide: cloning by PCR and expression of soluble, active protein in E. coli.

Ancylostoma caninum Anticoagulant Peptide (AcAP) is the major anticoagulant activity present in extracts of adult Ancylostoma caninum hookworms. This 8.7 kDa protein is a potent and specific inhibitor of human coagulation factor Xa. Using PCR, we have isolated a cDNA encoding for AcAP from an adult A. caninum cDNA library. The 5' end of the AcAP cDNA was identified by reverse transcription PCR (RT-PCR) using A. caninum cDNA and a 5' primer corresponding to a nematode spliced leader sequence. The AcAP cDNA was expressed in E. coli using a prokaryotic expression vector, and the recombinant fusion protein (rAcAP) was purified to homogeneity using nickel resin affinity chromatography and reverse phase HPLC. Purified rAcAP is comparable to the native protein in inhibitor activity, with an apparent equilibrium inhibitory dissociation constant (Ki*) for the inhibition of factor Xa of 265 +/- 71 pM. The purified protein also prolongs the prothrombin and partial thromboplastic times of human plasma in a dose dependent manner.

Use of specific immunoradiometric assay to determine preterm neonatal insulin-glucose relations.

Highly specific immunoradiometric assays were used to measure plasma concentrations of insulin, proinsulin, and 32-33 split proinsulin in neonates (n = 16). Neonatal plasma insulin concentrations were high relative to blood glucose concentrations and compared with adult insulin-glucose relations. Concentrations of proinsulin and 32-33 split proinsulin together accounted for 34-70% of the total concentration of insulin and pro-peptides. This study confirms the need to use a specific assay and neonatal reference data in the diagnosis of neonatal hyperinsulinism, and shows that neonatal pancreatic beta cell function may differ from that of older subjects.

Efficacy of an ivermectin/pyrantel pamoate chewable formulation against the canine hookworms, Uncinaria stenocephala and Ancylostoma caninum.

The effectiveness of the combination of pyrantel pamoate (5 mg kg-1) and ivermectin (6 micrograms kg-1) against the canine hookworms Uncinaria stenocephala and Ancylostoma caninum was determined. This combination is intended for monthly use as a heartworm preventative and for treatment and control of canine hookworms. The formulation was found to be effective (99.6% reduction in worm burdens) against both species of hookworms in experimentally infected dogs. No adverse effects due to the drug combination were observed in any dog during the course of this study.

Differentiation between the human hookworms Ancylostoma duodenale and Necator americanus using PCR-RFLP.

There are 2 major species of hookworms that infect humans. Ancylostoma duodenale and Necator americanus. Although traditionally considered to be identical for treatment purposes, there are significant life history differences between the species that must be considered for the rational design of chemotherapeutic and immunoprophylactic control strategies. However, identification of the species infecting a particular population has been problematic, as the eggs of the 2 species cannot be reliably differentiated by classical parasitological methods. A PCR-based technique for the differentiation of hookworm species that infect humans is reported. A fragment of the 3' untranslated region of the cAMP-dependent protein kinase catalytic subunit gene was amplified from A. duodenale and N. americanus genomic DNA using primers derived from the corresponding A. caninum cDNA. Digestion of the amplified DNA with the restriction enzymes HpaII, MboI, TaqI, and ThaI generated specific restriction fragment patterns unique to each species. The technique can distinguish between pure and mixtures of hookworm DNA and can amplify DNA from a single egg. The primers also amplify the fragment from the DNA of several other species of hookworms that infect humans and other animals. The technique is fast, simple, and hookworm specific and represents a considerable savings in time over current methods used for distinguishing between human hookworm infections.

Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum.

Previous studies demonstrated that third-stage, developmentally arrested larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to canine serum and hostlike temperature. Experiments to determine the identity of the serum stimulus are described. Serum from several nonhost species stimulated feeding, but to levels lower than canine serum. Heating the serum to 57 C had no effect on its stimulatory ability. Dialysis reduced serum stimulatory activity by 50%, and ultrafiltration through 10- and 30-kDa molecular weight cut-off membranes decreased activity in both the filtrates and retentates similarly. Recombination of the filtrates and retentates restored activity to whole serum control levels. Commercial canine and bovine albumin stimulated feeding to serum control levels at 10 and 50 mg/ml, respectively. These results suggest that albumin and an unidentified low molecular weight compound(s) are capable of inducing in vitro feeding by A. caninum L3.

Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum.

Developmentally arrested nonfeeding infective larvae of hookworms resume development after entry into the host, presumably in response to a signal encountered during invasion. Logically, an initial step in the resumption of development might be the resumption of feeding. An in vitro assay for feeding is described for the third-stage larvae of the canine hookworm Ancylostoma caninum. Populations of larvae incubated under hostlike conditions in the presence of 10% canine serum resume feeding within 6 hr, as evidenced by the uptake of fluorescein-labeled bovine serum albumin. Feeding is dependent on the presence of canine serum, and peaks by 24 hr incubation. Maximal feeding levels occur at temperatures above 34 C with a gas phase of 5% CO2/95% air, whereas culture medium and pH are unimportant for feeding. Serum concentrations between 0.1% and 1.0% (v/v) initiate feeding, and the response peaks at approximately 8.0% serum. Serum triggers feeding within 6 hr and is not required for feeding to continue once initiated. The saturation effect and the trigger phenomenon suggest that the initiation of feeding is a receptor-mediated response.

Species-specific identification of human hookworms by PCR of the mitochondrial cytochrome oxidase I gene.

Significant differences in the life histories of the human hookworms Ancylostoma duodenale and Necator americanus necessitate their differentiation for epidemiological studies and the design of control programs. Current methods of identification require time-consuming, labor-intensive techniques. A polymerase chain reaction (PCR)-based method that enables rapid species identification is described. The mitochondrial cytochrome oxidase I genes of both species were sequenced, and species-specific primer sets were designed. The primers were used in PCR to amplify 585-bp fragments of the cytochrome oxidase gene from individual hookworm eggs, larvae, and adults. The technique was also able to identify mixed infections containing equal amounts of eggs from each species. The technique is rapid, technically simple, and sensitive and will permit the accurate identification of human hookworms in epidemiological field studies.

Resumption of feeding in vitro by hookworm third-stage larvae: a comparative study.

Third-stage infective larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to several stimuli. Experiments were conducted to characterize the in vitro feeding behavior of several hookworm species. Reduced glutathione and, to a lesser extent, canine and human serum stimulated third-stage larvae of Ancylostoma duodenale to resume feeding. Glutathione-induced feeding reached a maximum by 16 hr and was concentration-dependent between 0- and 15-mM glutathione. Oxidized glutathione and the reducing agents dithiothreitol and L-cysteine failed to induce feeding, suggesting that reducing conditions alone were not stimulatory. Serum incubated with glutathione was the most efficient stimulus for Ancylostoma ceylanicum, Ancylostoma braziliense, and Ancylostoma tubaeforme larvae, whereas Uncinaria stenocephala larvae responded best to canine serum alone. Necator americanus larvae did not resume feeding in response to glutathione, serum, glutathione plus serum, or linoleic acid (0.1-10 mM). These differences in feeding behavior suggest that generalizations concerning hookworm biology must be interpreted cautiously.

Variation between ASP-1 molecules from Ancylostoma caninum in China and the United States.

Hookworm infection continues to be a serious problem in rural areas of China. Rapid reinfection and high cost limit the effectiveness of deworming programs. Vaccination offers an attractive alternative to mass chemotherapy. However, variation in vaccine antigens from field hookworm populations could conceivably limit efficacy of a vaccine developed from laboratory strains. Reported here are initial experiments to ascertain levels of molecular variation in a promising vaccine antigen, ASP-1, from the dog hookworm Ancylostoma caninum. ASP-1 from a Chinese strain of A. caninum was isolated from a third-stage larval cDNA library and compared to ASP-1 from a U.S. strain. There was 97% and 98% similarity in the DNA and amino acid sequences, respectively. There were 42 polymorphic sites between the sequences, 30 of which were synonymous. The 12 nonsynonymous substitutions resulted in 10 changes in the deduced amino acid sequence. Five of the amino acid changes were in the N-terminal domain, whereas the C-terminal domain was more highly conserved, containing only 2 amino acid changes. The results suggest that the effect of molecular variation in antigens from geographically separated parasite populations should be considered during vaccine development.

Epidemiology of Necator americanus hookworm infections in Xiulongkan Village, Hainan Province, China: high prevalence and intensity among middle-aged and elderly residents.

Hookworm is highly endemic to Hainan Province, an island located in the South China Sea. To investigate the prevalence and intensity of infection in the area, the village of Xiulongkan was surveyed between April and July 1998. A cross-sectional study was conducted in which fecal samples of 80% of the village residents (631 individuals) were tested for the presence of helminth eggs. Hookworm was the predominant intestinal helminth in Xiulongkan, where it was determined that 60% of those tested were infected. Necator americanus was the predominant species of hookworm in this population. The prevalence of hookworm increased with age, and then leveled to a plateau for ages 41 yr and up. This observation was in contrast to infections with Ascaris lumbricoides, where the highest prevalences occurred among school-aged children. Women had a significantly higher prevalence of hookworm than men and this difference emerged in early adulthood. The intensity of hookworm infection also significantly increased with age, with the highest intensity infections occurring among middle-aged and elderly residents. Females were more likely to have moderate or heavy infections, whereas males were more likely to have light infections. The rates of hookworm transmission are particularly high among the middle-aged and elderly residents of Xiulongkan.

Neonatal hypoglycaemia--blood glucose monitoring and baby feeding.

Recent concerns regarding neurological sequelae of neonatal hypoglycaemia have raised the question of whether demand breast feeding may increase the risks of neonatal hypoglycaemia and neurological handicap. In this review article neonatal hypoglycaemia is defined, monitoring of babies for this condition is discussed and implications for baby feeding practices are stated.

Epidemiology of human intestinal nematode infections in Wujiang and Pizhou counties, Jiangsu Province, China.

Intestinal nematode infections are considered highly endemic in the Chinese province of Jiangsu. In May 1997, the prevalence of intestinal nematodes infections was determined among all of the inhabitants aged 5 to 65 of the southern Jiangsu village of Yaojiakon (Wujiang County) and the northern Jiangsu village of Jianmiao (Pizhou County). It was determined that the prevalence of ascariasis, trichuriasis and hook worm infection was greatly reduced compared to when it was last measured in 1990. The reduction was noted to be particularly impressive in Yaojiakon village where the prevalence of ascariasis and trichiuriasis fell to 2% and 1.5% respectively. Much of this reduction was attributed to improvements in economic development which have occurred in southern Jiangsu Province at a rapid pace. In contrast, the reduction in nematode infections among villagers living in northern Jiangsu was more modest. The most striking reduction in both villages was in school-aged children who since 1988 have received yearly treatments with anthelminthic drugs. The intensity of nematode infections was investigated for hookworm where 70% of hookworm-infected Yaojiakon villagers were found to harbor light infections (< 400 eggs per gram) compared to 83% of hookworm-infected Jianmiao villagers. Necator americanus was the predominant hookworm in Yaojiakon village (South), whereas Ancylostoma duodenale predominated in Jianmiao village (North). The majority (76%) of hookworm-infected patients developed IgG antibodies against N. americanus antigen, although 20% of uninfected patients living in the village also had circulating antibodies. Intestinal nematode infections continue to be a significant public health problem in Jiangsu Province although their prevalence has decreased since 1990.

Epidemiology of human ancylostomiasis among rural villagers in Nanlin County (Zhongzhou village), Anhui Province, China: age-associated prevalence, intensity and hookworm species identification.

Hookworm infection has traditionally been highly endemic to Anhui Province, China. Zhongzhou village in southwestern Anhui was identified as an endemic focus of infection caused by the hookworm Ancylostoma duodenale. This species was found to predominate over Necator americanus in ratios ranging from 35:1 based on the recovery of third-stage infective larvae (L,) to 21:1 based on the recovery of adult hookworms after anthelmintic chemotherapy. The overall prevalence of Ancylostoma infection in Zhongzhou was 33.2% with a greater prevalence among males. Unlike the age-associated prevalence patterns for N. americanus in Hainan and other southern Chinese provinces that show increasing prevalence with age and the highest prevalence among the elderly, the age-associated prevalence for A. duodenale in Zhongzhou exhibited a peak in middle-aged adults with subsequent decline. The age-associated intensity pattern exhibited a similar trend although the most of the hookworm infections were light or moderate infections as defined by quantitative egg counts.

[Sequencing of cytochrome C oxidase 1 gene of Ancylostoma duodenale and Necator americanus].

AIM: To identify the genetic diversity between Ancylostoma duodenale and Necator americanus. METHODS: Mitochondrial cytochrome C oxidase subunit 1 (CO1) gene was amplified from genomic DNA of human hookworms collected from infected patients in Hejiang County, Sichuan Province, and the purified PCR products were directly sequenced by using Licor auto-sequencer. RESULTS: The PCR products were about 700 bp. Alignment of CO1 gene fragment sequences showed 89.7% similarity between Ancylostoma duodenale and Necator americanus, but still certain nucleotide variations (10.3%) existed. CONCLUSION: CO1 gene sequence can be used as a marker to identify the two species of human hookworms.

Babies born after diabetes in pregnancy: what are the short- and long-term risks and how can we minimise them?

Despite improvements in services for people with diabetes and an increased focus on care of diabetes in pregnancy, there has been no significant reduction in neonatal complications after pregnancy complicated by maternal diabetes. Some complications are severe and life threatening or lead to long-term difficulties, whilst others are transient and are unlikely to lead to long-term harm, if managed according to standard guidelines. Most neonatal complications are, in theory, avoidable by optimal diabetes care, those that arise directly as a result of poor control of diabetes in pregnancy or as a result of obstetric interventions related to maternal diabetes control. Of greater concern are iatrogenic complications that arise from decisions which have no clear rationale (e.g., 'routine' admission of a baby to a neonatal unit). Planning for neonatal management must take into account known risks and the likelihood of occurrence, start in advance of delivery, involve all relevant groups of professionals and be centred on the needs of the mother and baby and not upon historical organisational policies.