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.

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.

Tegumental changes in adult Schistosoma mansoni harboured in mice treated with praziquantel enantiomers.

Praziquantel administered to the host causes damage to the tegument of Schistosoma mansoni. In this study, the effects of racemic praziquantel (Pra) and its enantiomers, levo-praziquantel (L-Pra) and dextro-praziquantel (D-Pra) were compared using scanning electron microscopy (SEM). Mice infected with S. mansoni for 49 days were treated with a single dose of Pra (300 mg/kg), L-Pra (150 mg/kg) or D-Pra (150 or 600 mg/kg). Groups of three mice were killed after 4 and 24 h, and schistosomes collected by perfusion and examined by SEM. Treatment with Pra or L-Pra, for 4 or 24 h, caused tegumental damage to S. mansoni including severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the tubercles, collapse and peeling. After treatment with D-Pra at 150 mg/kg, no apparent damage was observed. When the dosage was increased to 600 mg/kg, after 4 h lesions on the tegument similar to those induced by Pra or L-Pra were seen, but less severe. After 24 h, there was evidence of recovery. The study thus clearly showed that L-Pra was more active than D-Pra in causing tegumental damage. D-Pra showed a qualitatively similar activity at a higher concentration. It is possible that this effect was due at least to some extent to the small amount of L-Pra (<2%) which was present in the preparation of D-Pra used.

Preventive effect of artemether in experimental animals infected with Schistosoma mansoni.

The effect of artemether, an antimalarial drug developed from the plant Artemisia annua, has been tested against the larval stages of Schistosoma mansoni covering the time from skin penetration to the early adult liver-stage. The results show that the experimental animals used (hamster and mice) do not develop schistosomiasis mansoni if treated with artemether during the first month after infection. The parasite was found to be especially susceptible between the 3rd and 4th week after infection, resulting in worm reductions of 75.3-82.0% compared to non-treated controls. This level was boosted to 97.2-100% when the animals were subjected to various schedules of repeated treatment. Almost complete protection was also reached in parallel experiments with repeated infections carried out to mirror more closely the real situation of trickle infection.

Effect of praziquantel together with artemether on Schistosoma japonicum parasites of different ages in rabbits.

The efficacy of combined treatment with praziquantel and artemether against infection with Schistosoma japonicum was tested on infected rabbits, in which 7-to 14-day-old schistosomules and 42-day-old adult schistosomes were simultaneously present. Rabbits were treated orally with praziquantel and artemether using various dosages and schedules. The therapeutic effects were evaluated by estimating the mean total worm burden (TWB) and female worm burden (FWB) and comparing them with the worm burdens in control animals treated with praziquantel or artemether alone. When the rabbits received praziquantel in a single dose (50 mg/kg), or daily for 2-6 days (30-60 mg/kg), the TWB was reduced by 28-66% and the FWB by 26-65%. In rabbits treated with artemether the reductions were 44-56% and 35-54%, respectively. Treatment with praziquantel in combination with artemether resulted in a significantly greater reduction of worm burden than was found for the groups treated with praziquantel or artemether alone, using the same dosages and schedules. TWB was reduced by 79-92%, and FWB by 80-93%. The results demonstrated that when rabbits infected simultaneously with schistosomules and adult schistosomes were treated with praziquantel in combination with artemether, the effects of the individual drugs could be increased significantly.

Epidemiology of human geohelminth infections (ascariasis, trichuriasis and necatoriasis) in Lushui and Puer Counties, Yunnan Province, China.

Between April and June of 1998, the prevalence and intensity of geohelminth infections caused by hookworm, Ascaris and Trichuris were investigated in two rural Yunnan villages. In Liuku, a village of Lisu indigenous people in Lushui County, there was an overall geohelminth prevalence of 72% (48%, 43% and 16% for hookworm infection, ascariasis, and trichuriasis, respectively). The prevalence of ascariasis was greatest among preschool and school aged children, whereas the prevalence of trichuriasis was greatest among teenagers and the prevalence of hookworm increased until the age of 10-15 and then remained high throughout adulthood. In Linger, a village of Han Chinese, located in Puer County, there was an overall geohelminth prevalence of 77% (30%, 60% and 36% for hookworm infection, ascariasis, and trichuriasis, respectively). The differences in prevalence for hookworm and ascariasis were statistically significant. The prevalence of hookworm in Linger increased steadily with age and did not plateau, but there were no discernible patterns of prevalence versus age for either ascariasis or trichuriasis. Heavy trichuriasis infections were noted to occur in Linger. In both villages, more than 98% of the hookworm infections were of light and moderate intensity. Both by morphologic identification of third-stage infective larvae (L3) from eggs as well as identification of adult hookworms recovered from adult residents after treatment with quantrel, Necator americanus was identified as the exclusive hookworm in each village. Geohelminth infections caused by Ascaris, Trichuris and hookworm remain highly endemic to the rural areas of Yunnan Province in southwestern China.

Artemether, an effective new agent for chemoprophylaxis against shistosomiasis in China: its in vivo effect on the biochemical metabolism of the Asian schistosome.

Conventional drug chemotherapy against human schistosomiasis currently relies on treatment with praziquantel to eliminate adult schistosome worm pairs. The use of praziquantel for control purposes is limited, however, by high rates of post-treatment re-infection with subsequent parasite egg deposition and host end-organ damage. Artemether, a methyl ether derivative of the anti-malarial drug quinghaosu, was discovered recently to also have anti-schistosomal properties. Because artemether selectively targets the larval migratory stages of the parasite, known as schistosomulae, it blocks the development of ovipositing adult schistosome worm pairs in the vasculature. On this basis, we have since shown in clinical trials conducted in China that artemether has proven benefit as an agent for chemoprophylaxis. In vivo studies using laboratory animals suggest that artemether causes damage to the tegument and musculature of schistosomulae. Artemether may exert its helminthotoxic effect through synergy with hemin or related heme-containing compounds. Schistosomes recovered from artemether treated laboratory animals have increased glycogen phosphorylase activity, but decreased glucose uptake. These findings may account for their decreased glycogen content, relative to schistosomes recovered from untreated laboratory animals. The artemether-damaged schistosomes also have decreased activities of a number of enzymes and enzyme systems, including glycolysis. This might suggest common pathways by which artemether may target human parasites that live in the bloodstream.

Epidemiology of human ancylostomiasis among rural villagers in Nanlin County (Zhongzhou Village), Anhui Province, China: II. Seroepidemiological studies of the age relationships of serum antibody levels and infection status.

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.

Hookworm burden reductions in BALB/c mice vaccinated with recombinant Ancylostoma secreted proteins (ASPs) from Ancylostoma duodenale, Ancylostoma caninum and Necator americanus.

Vaccination of mice with alum-precipitated recombinant Ancylostoma secreted protein-1 from the canine hookworm Ancylostoma caninum (Ac-ASP-1) results in protection against A. caninum larval challenge. Vaccine protection is manifested by host reductions in hookworm burden compared to control mice. The goal of this study was to determine whether ASP antigens cloned and expressed from different hookworm species will cross protect against A. caninum larval challenge. Cross-species protection against A. caninum challenge infections was observed with immunizations using recombinant ASP-1 from the human hookworms Ancylostoma duodenale and Necator americanus. However, the degree of protection was proportional to the extent of amino acid sequence homology between the ASP immunogen used for vaccination and the Ac-ASP-1 produced by the challenge larval strain. Vaccine protection was noted to decrease significantly as amino acid sequence homologies diverged 10% or more. It was also determined that Ac-ASP-2, a molecule cloned from A. caninum having 55% amino acid sequence homology to the C-terminus of Ac-ASP-1, did not elicit vaccine protection. These observations were partly reflected in the titer of antibodies that recognize Ac-ASP-1. The studies reported here will help to design immunogenic peptide vaccines based on the sequence divergence of hookworm ASPs.

Experimental approaches to the development of a recombinant hookworm vaccine.

Hookworm infection is a major parasitic cause of morbidity in the developing nations of the tropics. Development of a genetically engineered vaccine would be a useful tool in the control of this infection in highly endemic areas. Recombinant polypeptides belonging to the Ancylostoma secreted protein (ASP)-1 family have shown promise for reducing hookworm burdens after larval challenge infections in mice. Typically, these polypeptides are expressed in Escherichia coli and administered as an alum precipitate. Vaccine protection is antibody dependent. It is anticipated that a cocktail of different recombinant hookworm antigens may be required in order to effectively prevent heavy hookworm infections and disease. The progress of this work has been hampered by the absence of both a convenient laboratory animal with which to study hookworm infections resembling human infection, as well as the lack of easy availability of native hookworm antigens. In addition, useful human serologic correlates of antihookworm immunity are still poorly defined.

Epidemiology of human hookworm infections among adult villagers in Hejiang and Santai Counties, Sichuan Province, China.

Hookworm infection as well as other intestinal nematodiases are endemic to Sichuan Province in China. In order to research the prevalence and intensity of these infections we visited two villages in Hejiang County (southern Sichuan Province) and Santai County (northwestern Sichuan Province) between July and October of 1997. Fecal examinations were performed on adult villagers over the age of 15 years (currently children under this age are dewormed annually with anthelmintic drugs). Among 310 residents of Lugao Village (Hejiang County), 87, 63 and 60% were infected with hookworm, Ascaris or Trichuris, respectively. The prevalence of hookworm determined to rise linearly with age (r = 0.97). High intensity infections with hookworm still occur in this region as 22% of the residents have over 3000 eggs per gram (PEG) of feces as determined by quantitative egg counts. The majority of these individuals harbored mixed infection with Necator americanus and Ancylostoma duodenale, although the former predominated when adult hookworms were collected from 30 village residents treated with pyrantel pamoate. In contrast, among the 334 Xinjian villagers examined (Santai County) the majority harbored predominantly light hookworm infections--66.1% of the residents has less than 400 EPG by quantitative fecal examination and only 3.7% exhibited greater than 3000 EPG. Again, N. americanus was the predominant hookworm seen after worm expulsion. We have round that despite economic development which is occurring in some parts of China, significant hookworm infections and clinical hookworm anemia still exist in areas of Sichuan Province. In Hejiang County we found that the intensity of hookworm infection has actually risen within the last 10 years. Hookworm is a medical problem among the elderly in Sichuan.

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.

Histochemical alterations of infective third-stage hookworm larvae (L3) in vaccinated mice.

To study the histochemical alterations of hookworm L3 administered in a challenge dose to mice vaccinated previously with the larvae. Male Kunming strain mice vaccinated subcutaneously with 500 living Ancylostoma caninum L3 once every 2 weeks for a total of three immunizations before a final challenge with 500 L3 one week after the final immunization. The abdominal skin with underlying subcutaneous tissue and muscle were removed from the site of percutaneous challenge entry (from 2-3 mice), and fixed in absolute alcohol, cold acetone and 10% neutralized formalin. The tissue sections containing the L3 from the challenge dose were then stained histochemically of glycogen, RNA, DNA alkaline protein, acid mucopolysaccharide, collagen, reticulin, alkaline phosphatase (AKP) and adenosine triphosphatase (ATPase). Skin samples from non-immunized mice that were also subcutaneously inoculated with the L3 served as negative control. The L3 identified in cutaneous sections from vaccinated mice at 6-72 hours post-challenge exhibited reductions in parasite glycogen, alkaline protein, RNA and DNA, as well as reductions in acid mucopolysaccharide, collagen and reticulin contents in the parasite cuticle. There were also reduced enzyme AKP and ATPase activities. In contrast L3, identified in sections from non-immunized mice exhibited a normal histochemical appearance, as did some L3 who survived in vaccinated mice at 7-14 days post-challenge. Vaccination results in hookworm L3 damage which is manifested by reduced histochemical staining for the challenge inoculum of parasites. There is also reduced hydrolytic enzyme activity. The observed changes could reflect either host-mediated parasite structural damage and disintegration or possibly anti-metabolic properties of the host immune response.

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.

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.