Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

Anemia severa causada por Necator americanus: reporte de un caso / Severe anemia caused by Necator americanus: case report

En climas templados, yungas (zona intermedia entre valle y trópico) y trópico, prevalecen dos parásitos hematófagos: Necator americanusy Ancylostoma duodenale, responsable de una anemia crónica, que lleva a los niños a tener niveles tan bajos de Hb, que a veces sonconsiderados incompatibles con la vida; rara vez originan sangrado agudo severo.Describimos el caso de un niño, de seis meses de edad, procedente de Coroico (Nor Yungas) con disentería inicial, misma que luego secomplica con melenas y rectorragia de sangre rutilante que origina una hipovolemia severa, rayana en el choque. Sometido a exploraciónquirúrgica por persistencia del sangrado, previo gammagrafía para descartar un divertículo de Meckel, no se halla el sitio de sangradopor lo que se realiza laparotomía exploratoria y posteriormente una endoscopía, identifi cándose en duodeno foco de sangrado originadopor parásitos, que por su morfología se asume que se trate de Necator americanus. Con este diagnóstico, recibe transfusiones para suestabilizar su hipovolemia y Mebendazol, con buena evolución, remisión del sangrado de tubo digestivo, y buena tolerancia a la alimentación.El “sangrado agudo” por uncinarias, es excepcional, hecho que justifi ca la presentación del caso y permite realizar un recordatorio breve delos mecanismos responsables de ello...

In temperate climate, yungas (intermediate zone between valley and tropical lowland), two blood sucking parasites are prevalent: Necator americanus and Ancylostoma duodenale, responsible for chronic anaemia in children that causes extremely low Hb levels sometimesconsidered as incompatible with life; only in rare cases acute bleeding occurs.We describe the case of a six months old child stemming from Coroico (Nor Yungas) initially with dysentery that becomes complicated by melaena and rectal bleeding originating severe hypovolaemia bordering on shock. After gammagraphy in order to exclude Meckel’s diverticle, the child was submitted to surgical exploration, where no source of bleeding could be found, therefore an explorative laparotomy was performed and later on an endoscopy, where the source of bleeding was identifi ed in the duodenum as caused by the parasite Necator Americanus. With this diagnose, blood transfusions were administered to the child in order to stabilize the hypovolaemia and Mebendazol was given. The evolution was good, the bleeding stopped and feeding was well tolerated.Acute bleeding caused by hook worms is exceptional; therefore we present this case as a short reminder of the responsible mechanisms.

Necator americanus in the DSN hamster: density-dependent expulsion of adult worms during primary infection.

Neonatal hamsters were exposed to varying doses of Necator americanus larvae and changes in the stability of the resulting worm burdens were monitored over a period of 25 weeks. No change in worm burdens was evident for the first 5 weeks of infection, irrespective of the infection intensity, but the more heavily infected groups subsequently lost worms in a density-dependent manner. Male and female hamsters lost comparable proportions of their established parasite burdens indicating that there was no host sex-linked difference in this respect. By week 15 infections had stabilized and the residual worm burdens, usually a maximum of 30 worms survived for a considerably longer period of time. Initially the percentage of male worms varied from 45% to 50% but as infection progressed male worms comprised a significantly increasing proportion of the total worm burden. By week 25 the percentage of male worms was usually in excess of 60%. The growth of infected animals was not severely affected by N. americanus, even when heavy worm burdens established initially, but a significant effect was detected particularly in week 5, prior to worm loss, when the adult worms would have been feeding on intestinal tissues and causing blood loss for a period of about 2 weeks. The most severe depression in the packed cell volume was also recorded in week 5, indicating that anaemia had been initiated in infected hamsters. Whilst, the regulation of parasite burdens in weeks 5-10 post-infection may have resulted from host immunity, the persistence of the residual worm burdens, the marked density-dependent anaemia and the subtle effect on host weight, all reflected well-documented aspects of chronic human necatoriasis.

Necator americanus in the mouse: histopathological changes associated with the passage of larvae through the lungs of mice exposed to primary and secondary infection.

The mouse/Necator americanus model was studied to assess the histopathological changes that occur in the lungs following primary and secondary exposure to infective larvae. Groups of BALB/c mice were infected percutaneously and killed on various days post infection. Parasite numbers were counted, the bronchoalveolar leukocyte response was quantified and histological sections of lung material were examined for evidence of host protective inflammatory reactions. An increase in the inflammatory infiltration was observed between days 5 and 9 in both primary and secondary infections but was considerably more intense in re-infected animals. This involved a marked change in the character of the infiltrate, particularly in the number of eosinophils that were recovered in lavage fluid. More worms were trapped in the lungs of challenged mice, as assessed through their inability to escape from lung material incubated in vitro. Overall, the results were found to be compatible with the development of acquired resistance to N. americanus and the expression of host protective immunity during the development of challenge-infection larvae in the lungs.

Inibiçäo da vida externa de larvas de ancilostomídeos e de Strongyloides stercolaris por substâncias vegetais / Inhibitory assays of larval stage of Ancylostomatidae and Strongyloides Stercolaris by vegetable substances

Os AA apresentam os resultados obtidos com a utilizaçäo de extratos vegetais de 24 espécies na inibiçäo do desenvolvimento larvar de ancilostomídeos e de Strongyloides stercoralis. As partes vegetais utilizadas na elaboraçäo dos extratos foram as mais diversas; os solventes utilizados foram o hexano e o álcool; destes, nove extratos alcoólicos e dois extratos hexânicos revelaram atividade inibitória do desenvolvimento larvar

Ancylostoma duodenale and Necator americanus: effect of temperature on egg development and mortality.

The development and mortality of the eggs of Ancylostoma duodenale and Necator americanus in distilled water were monitored over a range of temperatures between 15 and 35 degrees C. Egg demography was examined within the context of a four-parameter mathematical model of development and mortality. Over the range of temperatures studied, egg mortality (mu) was an increasing exponential function of temperature (T) measured in degrees Celsius. A single model adequately described the mortality of both species (In [mu] = 0.41*T-6.87). The minimum time (tau) to hatching was consistently less for A. duodenale (In [1/tau] = -(0.79 + 53.05*[1/T]) than N. americanus (In [1/tau] = -(0.99 + 53.05*[1/T]]. The hatching rate (sigma when t greater than tau) was an increasing function of time and temperature in both cases, but the precise functional relationship was species specific.

Light, long-lasting Necator infection in a volunteer.

A Necator infection produced by percutaneous exposure of a volunteer to three larvae was followed with periodic egg counts for 4 months beyond 18 years when passage of eggs in the feces ceased permanently. During the 2nd year of infection, there was unintentional exposure to two additional larvae. Egg counts per gram of feces (EPG) were approximately 1,000 during the 1st year, 1,500 over the next 5 years, and less than or equal to 200 over the final 3 years, during which time there were 6 periods of approximately 1 month each when Kato thick-smear examinations revealed no eggs. During 6 of the 1st 14 years, when egg counts were done by the standard direct smear method, up to 37% of the eggs were infertile. Based on the assumption that two female worms were present during the 1st year, three during the next 3 years or more, and only one during the final 3 years, the estimated output per female in this light infection was 500 EPG in the worm's prime of life, and less than 200 EPG in the final year of the 17 or 18 year life span.

Experimental and field studies on the viability of eggs, and the infectivity of third-stage larvae of the human hookworm Necator americanus recovered from the creek waters of the Niger Delta, Nigeria, and their epidemiological significance.

In a combined field and laboratory study, the eggs and 3rd-stage infective larvae of the human hookworm Necator americanus were recovered from the creek waters of the Niger Delta, Nigeria. They were tested for viability and infectivity. Eggs recovered from fresh water creeks showed a viability or hatching index of between 17.5 and 23.7%. Eggs recovered from brackish water showed a viability index of 18.8-21.4% in 'normal' cultures, and a maximum index of 6.3% when cultured in brackish water. The differences in the viability indices of these eggs were not significant (P greater than 0.05). Infective 3rd-stage larvae were recovered from both fresh creek water (205-258 L3/litre of water), and brackish water (45 L3/litre of water). Larvae hatched from brackish water eggs that were cultured in the brackish water were 20% infective by 3 days, but lost all infective potential by 7 days post-hatching. Larvae from fresh water eggs, cultured in fresh water and 'normal' laboratory cultures reached 50% infectivity in 3-5 days, losing potential infectivity in 11-15 days post-hatching. The infectivity patterns of larvae recovered directly from fresh and brackish waters did not differ significantly (P greater than 0.05) from the infectivity patterns of larvae recovered from laboratory cultures of eggs from all the experimental sources. Although larvae recovered directly from fresh and brackish waters survived in these respective media under laboratory conditions, there was a significant difference (P less than 0.05) in their survival patterns. The survival and infectivity rates of field larvae were considerably reduced over time relative to control larvae.(ABSTRACT TRUNCATED AT 250 WORDS)

Necator americanus: temperature, pH, light, and larval development, longevity, and desiccation tolerance.

The effect of incubation temperature and pH on the hatch rate of eggs of Necator americanus, and the desiccation tolerance of the resulting infective stage-3 larvae were investigated in the laboratory under controlled conditions. Hatching did not occur below 15 C and above 35 C. A 21% hatch rate was obtained at 15 C while a 10.6% hatch rate was obtained at 35 C. The highest hatch rate (93.7%) was obtained at 30 C. The optimum pH for hatching was 6.0, but the larvae did not reach the infective stage. Incubation temperature of the eggs affected the longevity and desiccation tolerance of resultant infective larvae. Larvae hatched at 30 C and maintained at 26 C under bright fluorescent light had a 50% survival time (S50) of 4 days. In the dark or shade, the S50 for larvae raised at 30 C was 5 weeks, while that of larvae hatched at 20 C was 7 weeks. Incubation temperature also affected the desiccation tolerance of larvae. Larvae developed at 20 C were more resistant to desiccation at various relative humidity values than larvae hatched at 30 C.

Antigen expression during development of the human hookworm, Necator americanus (Nematoda).

The accumulated and de novo synthesized antigens expressed by L3, L4 and adult Necator americanus, recognized by both the natural host, man, and the experimental host, the hamster, were identified by immunoblotting and immunoprecipitation analysis. Following infection of neonatal hamsters serum samples were taken on days 17, 35 and 117. Only serum taken 117 days after infection showed significant reactivity in immunoblotting experiments, recognizing adult epitopes of 30,000, 33,000, 48,000 and 69,000 mol. wt thereby suggesting that few accumulated antigens are shared between developmental stages. By contrast, immunoprecipitation analysis of metabolically labelled proteins suggested that L3 and in particular L4 larvae synthesize some antigens which comigrate with those synthesized and accumulated by adult worms. In addition, L4 larvae synthesize a 41,000 mol. wt excretory/secretory (ES) stage specific antigen. Parallel experiments using serum samples from infected humans, demonstrated that hamsters and man recognize many antigens of identical molecular weight. Notable in this respect are accumulated adult antigens of 30,000, 33,000, 48,000 and 69,000 and de novo synthesized antigens of 30,000, 33,000, 44,000, 46,000 and 69,000 mol. wt. Some individual human sera mainly recognized L3 antigens of 47,000-69,000 mol. wt in immunoblotting experiments whilst others simultaneously recognized adult epitopes. This differential recognition of developmental stages by individual human sera suggests that genetic or epidemiological factors are operative and warrants further study. Overall, these studies confirm the pronounced immunogenicity of Necator americanus in both man and an animal model and pave the way for analysis of the relevance of these antigens to field situations.

Necator americanus in neonatally infected hamsters. The time-course of infection and antibody response to the surface antigens of L4 and adult worms.

Neonatal hamsters were infected with a hamster-adapted strain of Necator americanus, and the time-course of infection was followed by worm and faecal egg counts. Parasite eggs were first recorded during the 6th week of infection, increasing rapidly thereafter to peak in weeks 7-10. Male hamsters excreted more eggs than females, but both sexes were equally susceptible to infection and harboured comparable worm burdens. Faecal egg counts declined from week 10 onwards and this was associated with a loss of worms from animals with heavy infections. Low level infections were stable over the first 114 d of infection but worm fecundity nevertheless still declined over this period. Both hamster sexes responded similarly to surface antigens on adult worms, the antibody levels rising from week 5 onwards to reach a plateau in weeks 6-7, which persisted until the experiments were terminated. The major antigens recognised on the surface of adult worms had molecular masses corresponding to 25 kDa, 32 kDa, a doublet with the heaviest polypeptide resolving at 46 kDa, and a triplet with the heaviest at 67 and 93kDa. In contrast L4 had only 2 major cuticular antigens resolving at 41 and 93kDa. The 93kDa molecule on L4 and adult worms may be antigenically related.

The growth and migration of Necator americanus following infection of neonatal hamsters.

Necator americanus was studied in neonatally infected hamsters in order to determine precisely the growth and migration of the parasite in this laboratory host. Most larvae stayed at the skin infection site for at least 48 hours following administration of larvae and the movement to the lungs commenced on day 3. There was no significant growth at the skin site or during the first two days in the lungs. 98% of the larvae were recovered from the lungs by day 6 and showed signs of some growth and development. Moulting larvae were seen in the lungs on days 7 and 8, but intestinal worms, which were first detected on day 7, were all L4 larvae. These worms were significantly longer than the lung stages and henceforth grew rapidly. Over 80% of the worms were recovered from the intestine on day 9, only small numbers of larvae persisting in the lungs until day 12. Moulting worms were observed in the intestine on days 17 to 21, after which growth continued and did not slow until about the fifth week. Small quantities of eggs were occasionally detected as early as day 34 and continuous egg production commenced in the seventh week of infection reaching a peak by about the 10th week.

Site of resistance to Necator americanus in hamsters.

Resistance to the development of human hookworm, Necator americanus was examined in 3- to 6-week-old young adult hamsters. Only 3% of N. americanus infective third stage larvae (NaL3) reached maturity in the intestines of young adults as opposed to as many as 60% in 2-day-old baby hamsters. This seemingly effective resistance prevailing in young adults was investigated in some detail. The skin, the first site of contact for the invading NaL3, was bypassed during the infection process. Completely in vitro exsheathed NaL3 (ExNaL3) were used, and young adult hamsters were infected parenterally, by-passing the skin. Even after exsheathing the larvae artificially before infection and by-passing the skin, no improvement was seen in the development of N. americanus in the intestines of young adults. Higher infection doses also did not increase the worm burden. Some of the factors limiting the development of parasites in young adults were examined. N. americanus were monitored in lungs and intestines during various intervals after infection. Similar parasite burdens were apparent in lungs of baby as well as young adult hamsters. In the intestines, a significantly lower burden of N. americanus was seen during various intervals in young adults compared to the baby hamsters. Moreover, N. americanus were expelled soon after reaching the intestine. This comparative monitoring revealed the intestine as the seat of resistance against the establishment of N. americanus in young adult hamsters.

Necator americanus in infant rabbits: complete development, humoral antibody, leukocyte response and serum protein changes following infection.

Necator americanus, originally isolated from man, developed completely in infant rabbits (RSG-1). This infection was serially passed in infant rabbits up to the 6th generation without using any immunosuppressant. Two day old infant rabbits demonstrated a high degree of susceptibility to N. americanus. Studies revealed the retention of worms in the intestine for more than 150 days during which egg production was high. In this experimental model the leukocyte, antibody and serum protein responses due to N. americanus infection were monitored. Results showed that eosinophil numbers rose significantly from day 28 to 170. A maximum was observed on day 80 (41%) in rabbits of the fourth generation. Antibodies to N. americanus were detectable in infected rabbits by counterimmunoelectrophoresis using third stage larval antigen (Rabbit strain). Generally, total globulins increased with decrease in albumin. Serum protein increase was associated with alpha-2, beta and gamma globulin components.

Necator americanus: population structure, distribution, and fluctuations in population densities of infective larvae in contaminated farmlands.

Observations have been made over a 15-month period on the frequency distribution of human faecal deposits and infective larvae of Necator americanus in a hookworm endemic area. The agestructure of infective larvae in a field population and their vertical distribution in soils were determined. These studies were undertaken to examine the hypothesis that hookworm transmission in the tropics is discontinuous and limited mainly to the rainy season. The distribution of larvae was found to be overdispersed statistically and in general much greater numbers of L3s were recovered during the rainy season. The degree of overdispersion was also reduced during this season, and there was a tendency for larvae to be confined to the surface/topsoil. The implications of these findings have been discussed in relation to human hookworm parasitism in endemic areas of the tropics.