Defective mucosal immunity and normal systemic immunity of Mongolian gerbils,Meriones unguiculatus,to reinfection with Strongyloides venezuelensis

The systemic and local protective activity of Mongolian gerbils was examined after re-infection with Strongyloides venezuelensis. Mongolian gerbils were unable to expel S. venezuelensis adult worms from the intestine for over ten weeks after a primary infection. Therefore, immune animals were prepared by treating with mebendazole four weeks after a primary infection and then they were challenged by different maturation stages of the parasite; subcutaneous inoculation with the infective larvae ( L₃₎ obtained by faecal culture, oral administration of L3 obtained from the lungs of rats three days after a primary infection, or oral implantation of adult worms obtained from the intestines of rats seven days after a primary infection. The results show that, although immune animals were highly resistant against challenge infection by subcutaneous inoculation with cultured L₃, they were unable to expel orally administered lung-recovered L3 nor orally implanted adult worms. Although potentiated mastocytosis was induced by challenge infections with lung-recovered L₃ and adult worms, all mast cells were formalin-resistant, heparin-containing cells and never seen in the epithelial layer. In spite of the defective protective capacity at the intestinal mucosa, circulating antibody production specific to S. venezuelensis adult as well as L3 antigen was positive. Therefore, the inability of Mongolian gerbils to expel S. venezuelensis adult worms from the intestine seems to be due to the defects of effector/regulator cells, presumably mast cells, but not due to immune unresponsiveness to parasite antigen.     

Infections of Brugia pahangi in conventional and nude (athymic) mice

AKR, BALB/c and CBA/Ca and T.O. mice were completely resistant to infection with third stage infective larvae of Brugia pahangi. Third, fourth and fifth stage worms transplanted from the peritoneal cavity of jirds into the peritoneal cavity of mice continued to develop. BALB/c mice were the most susceptible of the strains tested and adult worms were obtained after each type of transplanted infection. Congenitally athymic nude mice were much less resistant to transplanted worms and infective larvae developed to full maturity in most of them. Ten of 14 athymic mice infected by the intraperitoneal (ip) inoculation of infective larvae had microfilariae in their blood or peritoneal cavities. At autopsy a percentage recovery of adult worms of 0-38% (mean 11.1%) was obtained. Microfilariae were only found in the blood of 2 of 6 athymic mice infected by subcutaneous (sc) infection and at autopsy 0-19.1% (mean 6.1%) recoveries were obtained. The thymic littermates of the nudes were more resistant than those most of the other strains used.     

Induction of protective immunity in dogs to infection with Dirofilaria immitis using chemically-abbreviated infections

Four dogs were immunized against Dirofilaria immitis infection by a series of 3 larval infections which were each subsequently terminated by ivermectin treatment. Two control dogs received ivermectin treatment alone. Following the final ivermectin treatment, dogs were challenged with infective larvae by subcutaneous inoculation, both free and contained within diffusion chambers. Three weeks after larval challenge the chambers were removed and live larvae were enumerated. Seven months after challenge dogs were killed and necropsied to collect and count adult D. immitis. Chambers recovered from immunized dogs had 63% fewer larvae than chambers from control dogs. At necropsy, control dogs had a mean of 28.5 adult worms whereas the immunized animals had an average of 0.5 worms (range 0-2). Sera collected from immune dogs throughout the study had elevated antibody levels to third- and fourth-larval stage antigens. Significant levels of immune protection were achieved with this immunization regimen. The data suggest that a multiple-stage parasite killing occurs in immune animals. It was not possible to associate immune protection with any of the 5 antigen subsets.     

The survival of adult Litomosoides carinii transplanted into cotton rats previously injected with irradiated stage 3 larvae

Adult Litomosoides carinii were surgically removed from the pleural cavities of normal cotton rats which had been infected 35 days previously by the inoculation of normal stage 3 larvae. Groups of 20 such worms were then transplanted into either the pleural cavities of other normal, uninfected cotton rats or into the pleural cavities of cotton rats which had previously received, by subcutaneous injection, either 3 X 50 L3 which had been exposed to 40 krads Cobalt 60 irradiation or 1 X 50 L3 irradiated at 16.2 krads. Cotton rats vaccinated with irradiated L3 prior to transplant accepted transferred worms for at least 65 days whereas uninfected cotton rats soon encapsulated and destroyed the transferred worms. Microfilariae were detected in the pleural exudate and peripheral blood of vaccinated cotton rats but not in that of unvaccinated cotton rats.     

Reconstitution by bone marrow grafting of the defective protective capacity at the migratory phase but not at the intestinal phase of Nippostrongylus brasiliensis infection in W/Wv mice

After a primary infection by subcutaneous inoculation with the infective larvae (L₃) of Nippostrongylus brasiliensis, the intestinal worm burden was higher and expulsion was slower in W/W^v mice than in ^+/+ mice. When the course of infection was segregated into the migratory and intestinal phases, protection during the migratory phase examined by the larval recovery from the lungs and that during the intestinal phase measured by worm burden after intraduodenal implantation with adult worms were both defective in W/W^v mice. The higher susceptibility of W/W^v mice during the migratory phase was normalized by bone marrow re constitution. On the other hand, higher susceptibility of W/W^v mice during the intestinal phase, which was measured by worm burden 24 h after intraduodenal implantation of the larvae recovered from the lungs of rats, was not normalized by bone marrow grafting. Furthermore, slower expulsion seen in W/W^v mice after intraduodenal implantation with adult worms was not hastened by bone marrow reconstitution. These results indicate that the protective mechanisms against N. brasiliensis operating during the migratory phase and those during the intestinal phase were different in terms of bone marrow dependency and that nonmyeloid cells utilizing c-kit ligand/receptor system to express their functions are involved in the rnucosal defence against N. brasiliensis.     

Experimental transmission of Wuchereria bancrofti to monkeys.

Infective larvae of Wuchereria bancrofti from laboratory-raised Culex pipiens fatigans and Aedes togoi mosquitoes fed on human volunteers in Jakarta, Indonesia (J strain) and Kinmen Island, China (K strain) were introduced into Taiwan monkeys (Macaca cyclopis) by subcutaneous inoculation, by foot puncture, or by permitting infected mosquitoes to feed weekly on the monkeys. Some animals were splenectomized and others were treated with varying regimens of immunosuppressants. Necropsy was done on monkeys that died or were killed and the entire bodies were examined for worms. A total of 78 monkeys (43 males and 35 females) were exposed to infection and parasites were found in 29% of the females and 63% of males. In infections of 38 days or less worms were recovered from the testes of males and the pelt, carcass and lymph nodes of both sexes, but after 42 days of infection most worms were in the testes of males, and a few were recovered from lymph nodes and carcasses of females. Worms recovered at 8-11 days were third-stage, those found between 14 and 38 days fourth-stage, and ones found between 42 and 103 days were young adults. After 148 days most were adults and microfilariae were seen in the uteri of female worms at 160 days and later. The parasites continued to grow in size with time. Microfilariae were detected in the blood of nine monkeys between 8 and 18 months and the patent period varied from 5-21 months. Microfilarial densities were low and erratic, and periodicity could not be determined. The effectiveness of methods of administering infections and the value of various treatment regimens seem uncertain; monkey antilymphocytic sera, however, appeared to have some influence. Parasites were found in 36% of the Taiwan monkeys given the J strain and 54% of those given the K strain. A limited number of M. mulatta (3), M.irus (fascicularis) (3) and Aotus trivirgatus (4) were also given infective larvae and adult W. bancrofti were recovered from the testes of one male M. mulatta and one male M. irus; uterine microfilariae were found in one female worm from the latter monkey. A. trivirgatus were negative. Low numbers of infective larvae recovered from mosquitoes fed on patent monkeys were introduced intermittently into seven clean monkeys and one became microfilaremic between 11 and 17 months postinoculation.     

Strongyloides venezuelensis infection in Syrian golden hamster, Mesocricetus auratus, with reference to the phenotype of intestinal mucosal mast cells

Syrian golden hamster, Mesocricetus auratus, was found to be a moderately susceptible host for the intestinal helminth, Strongyloides venezuelensis. After infection by subcutaneous inoculation with 3000 infective larvae(L₃), about 20% of them became adult worms in the small intestine, and, after a stable infection up to day 20, adult worms were slowly and gradually expelled towards day 45. Before infection, mast cells in the jejunum were about 30/10 villus crypt units and over 80% of them were formalin-resistant and berberine sulphate-fluorescence positive. After infection with S. venezuelensis, the number of intestinal mast cells gradually increased with time and about a half of them were formalin-sensitive and berberine sulphate fluorescence-negative. Intraepithelial migration of mast cells was never seen before and after infection. Heterogeneity of mucosal mast cells in terms of granular proteoglycans was further confirmed by the determination of critical electrolyte concentration. In spite of the heterogeneity of proteoglycans, enzyme-histochemical study revealed that practically all mucosal mast cells of Syrian golden hamsters were positive for chymase but negative for tryptase. Mast cells in the skin and tongue were also positive for chymase but negative for tryptase. Together with our previous study on mucosal mast cells of other rodents, phenotypic variances of mucosal mast cells seem to be closely related to the protective capacity against the genus Strongyloides.     

Subconjunctival zoonotic onchocerciasis in man: aberrant infection with Onchocerca lupi?

In the past few decades, 10 cases of cryptic, zoonotic onchocerciasis, including two subconjunctival infections, have been reported in man. In the majority of cases, Onchocerca cervicalis, O. gutturosa or O. dewittei, which normally infect horses, cattle and wild boar, respectively, were responsible for the lesions. However, the taxonomic status of the parasites involved in the two subconjunctival infections, both of which were European, has never been unambiguously determined. In such infections, the acute phase appears to be characterized by conjunctivitis. A single, strongly coiled, immature, female worm was found incorporated in a large granulomatous nodule, in the ocular and peri-ocular tissues, in the chronic stage of each of the two eye infections. Several, patent, sporadic cases of subconjunctival O. lupi infection have recently been reported in dogs. In terms of the location of the worms, clinical signs and histopathology, these canine infections were very similar to those seen in the two human patients with eye infection. When the parasites recovered from human eyes were compared morphologically with the Onchocerca spp. infecting animals in Europe, they appeared to be most similar to O. lupi. Although O. lupi is normally a parasite of dogs, it may thus also be responsible for aberrant, zoonotic, subconjunctival infections in man.     

Successful development of Brugia pahangi in T-cell deprived CBA mice

CBA mice were thymectomized and treated with anti-thymocyte serum. Seven such mice were given 90--100 infective larvae of Brugia pahangi each by intraperitoneal (ip) injection and 5 given 99-100 larvae each by subcutaneous (sc) injection. From 62 days after infection 6 of 7 mice infected ip had microfilariae in their peritoneal cavities. Only one mouse infected by sc injection showed microfilariae in peripheral blood and this not until 98 days. At autopsy 5-45 adult worms were recovered from the ip infected mice. Only 2 od the 5 sc infected mice had adults and these only 3 each. No microfilariae or adult worms were detected in similarly infected unthymectomized CBA mice.     

不同日龄致倦库蚊对马来丝虫易感性的比较研究

不同日龄致倦库蚊分别经胸内接种马来丝虫微丝蚴后,比较观察了蚊虫体内微丝蚴的黑化率、(?)虫率以及感染性蚊虫的比率。接种后第3、4、5、9d解剖接种蚊虫,结果表明,1～2日龄蚊虫体内微丝蚴黑化率显著高于15～16日龄蚊虫,分别为84.1％和57.5％。三期幼虫率则相反,分别为1.5％和3.7％。黑化的微丝蚴主要分布在蚊虫腹部,占72.3％和69.1％。     

Western blot analysis of reactivity to larval and adult Strongyloides ratti antigens in mice

Mice were infected once, twice or many times with Strongyloides ratti infective larvae, and the parasite was allowed to complete its development. Other mice were infected many times with either infective larvae only, by aborting the infection with cambendazole, or with adult worms transferred by intra-oesophageal intubation. Sera from these animals were analysed by immunoblotting against SDS-PAGE separations of larval and adult worm water-soluble, deoxycholate-soluble, sodium dodecyl sulphate-soluble and excretory/secretory antigens. Minimal antibody responses were observed after primary and secondary infections. Mice exposed to multiple complete infections reacted strongly to both larval and adult antigens but greater responses were observed against the larval preparations. Stage-specific effects were noted in mice infected with larvae only or adult worms only. Mice exposed only to larvae reacted with larval antigens and to a minor degree to somatic adult worm antigens while those mice which were exposed only to adult worms failed to react with any of the antigen preparations. Some cross-reactions were found, however, as mice infected only with larvae displayed strong reactions against both larval and adult excretory/secretory products. These data demonstrate differences in sero-reactivity to infective larvae and adult worms and suggest that humoral immunity is induced by larvae migrating through the tissues and not by adult worms in the gut.     

旋毛虫成虫可溶性粗抗原对小鼠的免疫保护作用

本文应用人工感染的方法从大白鼠获得大量成虫。经冻融、超声粉碎、高速离心制备出成虫可溶性粗抗原。将不同剂量的抗原与等体积弗氏完全佐剂乳化后间隔1周共免疫小白鼠3次。最后一次免疫后间隔1周每鼠攻击感染130条感染性肌幼虫,攻击感染后1周剖检成虫、新生幼虫,攻击感染后35天剖检肌幼虫。试验结果表明,以每只小白鼠免疫200μg成虫可溶性粗抗原所诱导的免疫力最好,诱导成虫减虫率达94.65％,肌幼虫减虫率达95.60％。初步研究表明,旋毛虫成虫可溶性抗原是很好的免疫原。     

Ancylostoma ceylanicum: migratory behaviour in golden hamsters after oral and parenteral infection

The infectivity and migratory pattern of Ancylostoma ceylanicum infective larvae (L3) were investigated in hamsters infected by various routes. Following oral administration 40-70% of L3 attained maturity and there was no tissue migration. Following subcutaneous inoculation a small number (1-1.2%) of L3 attained maturity in the intestine after completing the broncho-oesophageal journey. Larvae which penetrated the skin also became adult in the intestine. Most of the larvae entering parenterally remained at the site of infection and in the tracheal region for more than 100 days without undergoing any development, other than desheathment. Those transmitted orally to naive hamsters developed in the normal way. Larvae inoculated parenterally into female hamsters were able to infect offspring in milk, but could not cross the placental barrier.     

The effect of 5-fluorouracil and 5-fluorocytosine on the development of the filarial nematodes Brugia pahangi and Dirofilaria immitis

5-fluorouracil and 5-fluorodeoxyuridine at 30 mg/kg body weight daily for four days inhibit microfilarial production in Brugia pahangi in the jird. Disruption of intrauterine embryogenesis was observed in treated female worms but the compounds were not macrofilaricidal or microfilaricidal under the conditions employed. 5-fluorocytosine possessed no filaricidal or embryostatic activity. The inhibition of microfilaria production by 5-fluorouracil was temporary and larval production was resumed within nine weeks. The compound also inhibited the development of B. pahangi and Dirofilaria immitis larvae in the mosquito Aedes aegypti, when administered to cages of mosquitoes as a 0.01 or 0.001% solution in a 10% aqueous sucrose solution on cotton wool wicks. The development of infective larvae of B. pahangi in the jird was inhibited by 5-fluorouracil.     

The migration and localization of Loa loa infective and fourth-stage larvae in normal and immunosuppressed rodents

The migration and localization of the human filarial parasite Loa loa in laboratory mice (BALB/c and Swiss) and jirds (Meriones unguiculatus) was investigated. The rodents, either left immunocompetent or immunosuppressed with hydrocortisone, were each inoculated subcutaneously or intraperitoneally with 50 or 200 infective, third-stage larvae (L(3)) of L. loa. Groups of the rodents were killed at various times post-infection, up to day 40, to enable histological studies and permit developing larvae to be recovered. Larvae survived and developed for only 1 week in the immunocompetent rodents but for a mean of 3 weeks in the immunosuppressed. Most of the larvae were found in the subcutaneous tissues (81.9%), peritoneal cavity (14.9%), pleural cavity (1.8%) or the lungs and heart (1.3%) and none was detected in the spleen, kidney, intestine, liver or pancreas. Localization of the larvae appeared unaffected by the site of inoculation, the rodent species or strain, or the dose of L(3) used. The recovery of larvae (as a percentage of the number inoculated) was better among the rodents inoculated with 50 L(3) each than among those given four times as many L(3). The results of the histological studies not only confirmed the presence of larvae in the subcutaneous tissue (72.5%), muscles (11.7%) and peritoneal and pleural cavities (7.8%) of the infected rodents but also revealed worms in the lymphatic vessels of the mesentery and spinal cord (7.3%). These results indicate that most L. loa L(3) inoculated into a mammalian host localize in the cutaneous sites and that only a small proportion of them might migrate, using the lymphatic system, into the internal organs. The observation of migrating L. loa larvae in the lymphatic vessel of the meningeal envelope of the spinal cord, albeit in an experimental host, may explain why, in areas where human loiasis is endemic, neurological manifestations occasionally occur in those with L. loa infections.     

Eye disease related to onchocerciasis: A clinical study in the Aratha-ú, Yanomami Tribe, Roraima State, Brazil

The prevalence of ocular lesions due to onchocerciasis was evaluated among residents of the Yanomami Tribe, in the northern Amazon, Brazil, an endemic area for onchocerciasis. 83 natives were submitted to an ocular examination including an external examination, biomicroscopy, intraocular pressure measurement, and a fundus examination. Clinical, parasitological and serological tests were carried out simultaneously. The population demonstrated a high prevalence of eosinophilia, skin microfilaria (55%) and onchocercal subcutaneous nodules (35%). A high prevalence of probable onchocerciasis related eye lesions was detected. Punctate keratitis (41%) and microfilaria in the anterior chamber (39%) were found as well as other probable onchocercotic lesions—chorioretinitis (7.2%) and anterior uveitis (6.0%). Other anterior eye lesions (corneal leucomas, conjunctival injection, lid nodules) occurred in 51% of the individuals. The anterior eye lesions were more prevalent than the posterior lesions. We did not find an association of glaucoma with onchocerciasis. The prevalence of these suggestive ocular lesions strongly correlates with the cutaneous nodules and eosinophilia, suggesting that skin nodules may be an indication for an eye examination. The present study provides evidence that significant infection and eye disease due to onchocerciasis persists in certain regions of Northern South America.     

Repeated infections of Brugia pahangi in the jird, Meriones unguiculatus.

Male jirds, Meriones unguiculatus, were subcutaneously inoculated in the groin with 1 to 5 doses of infective-stage larvae of Brugia pahangi at weekly or monthly intervals. When a dose of either 25 or 75 larvae or 4 weekly doses of 25 larvae were given, 15-16% of the larvae were recovered as adults approximately 4 to 7 months post inoculation. Only 8-10% of the larvae were recovered if 4 weekly or 5 monthly doses of 75 larvae each were given. After an inoculation of 75 larvae, 25% of the worms were recovered at 30 days. The 30 day-old population consisted of an average of 10 female and 8.8 male worms. Jirds previously inoculated with 4 weekly or 5 monthly doses were challenged with an additional 75 larvae 30 days prior to necropsy. An average of only 4.5 thirty day-old female worms were recovered in these cases, presenting a 55% decrease as compared to the single inoculation situation. There was some decrease in the mean length of female worms in multiply-inoculated jirds, but no difference in the mean lengths of the male worm population from singly or multiply-inoculated jirds was observed. No differences in prepatent periods or in patterns of microfilaraemia were observed in singly or multiply-inoculated jirds.     

Immunogenicity of homogenates of the developmental stages of Litomosoides carinii in albino rats

Homogenates of different developmental stages of the filarial parasites, L. carinii, emulsified in Freund's complete adjuvant have been examined for their efficacy in conferring immunity to the infection in the albino rats. The results revealed that sonicated preparations of microfilariae and infective larvae induce high resistance to the infection in the animals. In contrast, soluble antigens of adults and sonicated homogenates of adult males were ineffective to induce such resistance. Some resistance seen when sonicated adult worms of both sexes were used as immunogens appears to be due to the microfilarial antigen present in the extracts.     

Protective immunity to Brugia malayi larvae in BALB/c mice: potential of this model for the identification of protective antigens

Protective immune responses against the infective larvae of Brugia malayi have been demonstrated in BALB/c mice. Various factors governing resistance to reinfection have been examined to provide baseline data for use of this model in studies of immunoprophylaxis. Parasites that established following a primary infection survived for approximately 10 days, following which numbers declined rapidly to a low level. Resistance was evidenced by a more rapid clearance of secondary infection parasites. The degree of immunity expressed was not related to the route of administration of the initial infection (subcutaneous, intravenous, intramuscular, or intraperitoneal). However, both the level of resistance and the rapidity of its expression were dependent on dose, with as few as 2 larvae stimulating measurable immunity. Sensitization with living male or female adult worms, fourth stage larvae or microfilariae of B. malayi, or infective larvae of B. pahangi conferred substantial resistance to larval challenge. Significant levels of immunity were also induced by dead B. malayi larvae (46%) and their aqueous extracts (76%), but not with the corresponding insoluble fraction. We suggest that this experimental system is ideally suited to aid in the identification of putative protective antigens in brugian filariasis.     

Bancroftian filariasis in a Philippine village: entomological findings.

Bancroftian filariasis in an isolated Philippine village has been intensively investigated; this paper reports the entomological findings. Surveys were carried out six months apart in the driest and wettest months. Significant transmission was demonstrated only during the wet season. Aedes poicillius was the major vector of Wuchereria bancrofti. Ae. poicilius accounted for 58% of larvae found in the axils of banana plants and 31% of those in abaca axils; negligible numbers of larvae of this species were found in pandanus and gabi axils. Mosquitoes were collected from indoor harbourages twice weekly for five weeks during the wet season; 615 mosquitoes were caught of which 80% were Ae. poicilius and 9% were Culex quinquefasciatus. 11% of the former and 13% of the latter contained filariae; whereas all stages of development were seen in Ae. poicilius, no development beyond the first stage was seen in Cx. quinquefasciatus. Human bait trapping was used for 110 manhours; 371 mosquitoes were caught of which 58% were Ae. poicilius and 24% were Culex summorosus. Filarial larvae were seen only in Ae. poicilius; 3.7% of mosquitoes were positive and all stages of filarial development were seen. The mean landing/biting rate between 1900 and 0500 hours was 3.37 mosquitoes per man-hour with a maximum of almost seven mosquitoes per man-hour at midnight. Overall, 2.26% of vectors collected in the human studies were infective. There was an average of 3.38 third-stage larvae per infective mosquito. The efficiency of transmision was estimated as 6.1 x 10-5, or one new case of microfilaraemia for every 16, 400 bites by infective mosquitoes in the village population. In contrast to the human studies, large numbers of mosquitoes were caught by animal bait trapping in both the wet and dry seasons. The distribution of mosquito species was similar in the two seasons. Ae. poicillius represented only 1.0-1.5% of all mosquitoes seen. No filarial larvae were seen. It was concluded that transmission of filariasis in the village was inefficient and postulates were advanced to explain the increased intensity and severity of filariasis inmen as compared to women.