Nonenzymatic isolation of Trichinella pseudospiralis infective L1 larvae at pH 7.4

Over half of the number of Trichinella pseudospiralis infective L1 larvae recovered from host carcasses by pepsin-HCl digestion were isolated from homogenized carcasses incubated in HBSS. More worms isolated by the latter method were viable compared to those isolated by pepsin-HCl digestion. When host carcasses infected with T. pseudospiralis were diced into pieces and incubated in HBSS, 30% more worms were recovered than from homogenized carcasses incubated in HBSS as above, and the majority of worms acquired by the former method were viable. The infectivity of T. pseudospiralis infective L1 larvae isolated from homogenized muscle in HBSS was 3.9 times greater than that for larvae recovered from homogenized carcasses by pepsin-HCl digestion. Only 4% and 0.8% of the number of T. spiralis recovered from homogenized muscle by pepsin-HCl digestion were isolated from homogenized or diced muscle incubated in HBSS, respectively. Fewer T. spiralis isolated from homogenized tissue in HBSS were viable compared to those recovered from homogenized carcasses digested in pepsin-HCl or diced carcasses incubated in HBSS.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Dictyocaulus viviparus: Migration in agar of larvae subjected to a variety of physicochemical exposures

Dictyocaulus viviparus larvae were exposed to ox bile and CO₂ at intervals during their cultivation to the infective stage. Preinfective and young infective larvae were stimulated by CO₂. Bile slightly inhibited preinfective larvae, but stimulated the infective stage. Old coiled, resting infective larvae were stimulated by bile down to a concentration of 10 ppm of bile dry matter, by vertebrate biles of pig, sheep, newborn calf, cow, guinea pig, dog, and chicken, as well as by defatted bile dry matter and by glyco-, tauro-, glycodeoxy-, and taurodeoxycholates. Continuous bile exposure appeared necessary to maintain high larval activity. A high pCO₂ as well as a low redox potential potentiated the effect of bile, but had no effect alone. Exposure to pepsin-HCl and to trypsin had only a minor stimulatory effect.     

Alterations in the longevity and fecundity of adult Trichinella pseudospiralis related to method of isolation of infective larvae

The infectivity of Trichinella pseudospiralis infective larvae was reduced significantly following exposure to low pH or a combination of 1% pepsin at low pH compared to that for larvae isolated in phosphate-buffered saline (PBS) at pH 7.0. Reduction of host gastric pH by administration to mice of sodium bicarbonate solution in PBS was accompanied by an increase in the infectivity of larvae isolated in 1% pepsin/HCl (P/HCl) compared to that for worms inoculated into hosts given PBS alone. Fewer adult worms developing from larvae isolated in P/HCl became established in the host small bowel than was seen with larvae isolated in PBS; moreover, the fecundity in vitro of adult worms developing from P/HCl-isolated larvae was reduced below that for adults developing from larvae isolated from host muscle in PBS. More adult worms were recovered following infection of immune hosts with PBS-isolated larvae than were recovered from immune mice challenged with larvae isolated in P/HCl. Similar findings were observed in mice immunized by infection with Trichinella spiralis and challenged with T. pseudospiralis larvae isolated in either P/HCl or PBS. Immunization of mice with T. pseudospiralis larvae isolated by either method and challenged with larvae of T. spiralis resulted in recovery of similar percentages of the challenge inoculum.     

Strongyloides ratti: Acquired resistance in the rat to the preintestinal migrating larvae

Potential sites for expression of acquired resistance to Strongyloides ratti larvae in rats were investigated. In rats immunized by exposure to a single live infection and challenged 30 to 40 days later, 46 to 98% of the challenge larvae failed to reach the small intestine. Multiply immunized rats nearly completely eliminated migrating challenge larvae. This early killing of migrating larvae occurred during the first 48 hr after challenge infection. Resistance to migrating challenge larvae was also induced by repeated injections with heat-killed infective larvae. That the intestine may also serve as an effective site for worm expulsion was confirmed by intestinal transfers of worms from rats with primary infections into resistant rats.     

Immunization with infective larvae of Strongyloides ratti (Nematoda) exposed to microwave radiation

Microwaves have not been tested previously for possible application in producing immunogenic preparations of parasites. This study examines the immunizing capacity of microwave-irradiated, infective larvae of Strongyloides ratti in rats. Rats were inoculated subcutaneously with untreated, microwaved, or microwaved and homogenized larvae, or distilled water, and challenged with untreated larvae. Data were collected on egg production and worm number/rat during primary infections and on egg production, worm number/rat, worm size, and eggs in utero/worm following challenge. Our results demonstrated that microwaved, infective larvae (intact or homogenized) of S. ratti were immunogenic for rats, even though they were incapable of reaching the intestine and maturing to adult worms. The immunity elicited by exposure to microwaved larvae was characterized on challenge by a significant reduction in the number of eggs produced/worm, by the formation of perioral plugs, and by reductions in worm numbers and size. These results suggest that microwave radiation may provide a valuable new tool for parasitic vaccine production. In addition, we have demonstrated the occurrence of a feature of the immune response of rats to S. ratti that may have been overlooked previously; i.e., a gut-level response that was elicited by larvae, but manifested against adult worms in the intestine.     

Strongyloides stercoralis: oral transfer of parasitic adult worms produces infection in mice and infection with subsequent autoinfection in gerbils.

Oral transfer of parasitic adult Strongyloides stercoralis produced patent infections in gerbils, C57BL/6J and SCID mice. In gerbils receiving adult worms, 7.3% of the transferred worms established and autoinfective L3 were found beginning on day 5 post-transfer, with peak numbers seen on days 6 and 7 post-transfer and few seen by 9 days post-transfer. These results suggest that development of autoinfective L3 in the gerbil is limited by the immune response of the host. When given orally to mice, between 7.2% (C57BL/6J) and 19.5% (SCID) of the adult worms established. These levels are higher than those previously obtained by the subcutaneous infection of SCID mice with infective larvae. No autoinfective larvae were found in infected mice and the ratio of L1/adult worms was small compared with that seen in gerbils. Thus, mice infected orally can be used as a model to study the interaction between the adult worm and the host, and since autoinfection has not been seen in the murine model, as developed to date, orally infected mice may be useful as a model to study mechanisms preventing autoinfection.     

Effect of gamma-irradiation on the survival and development of the infective larvae of the hookworm, Gaigeria pachyscelis

Irradiation of the infective larvae of Gaigeria pachyscelis Railliet and Henry, 1910 with gamma rays upto 160 Kr had no significant effect on the in vitro survival of these larvae for a period of 49 days, maintained either at room temperature (32.2--39.4 degrees C) or at 4 degrees C. However, the behaviour of the irradiated larvae in the lamb host was much changed, as shown by a marked reduction in the worm establishment and the development of stunted and sterile worms from these larvae. As the level of irradiation increased, there was a corresponding decrease in the subsequent worm establishment. It was found that male larvae are more sensitive to the effects of irradiation than female ones, particularly at higher levels.     

Density-dependent effects on the survival and growth of the rodent stomach worm Protospirura muricola in laboratory mice

The spirurid nematode, Protospirura muricola, is of intrinsic interest as a rodent model of gastric nematode infections. Since worm burdens can be very heavy in nature, density dependent processes may constrain parasite growth. Laboratory mice (BKW) were exposed to varying doses of infective larvae of P. muricola in the range 5 to 40 third-stage larvae (L3), in four separate experiments in which progressively higher doses were utilized. All mice were culled 60 days after infection and a total of 518 worms (226 male and 292 female worms) was recovered, measured and weighed. Overall survival was 58.9%, but survival declined significantly with increasing dose by approximately 21% (from 66% at 5 L3 per mouse to 52% at 40 L3 per mouse). The length and weight of worms correlated positively in both sexes. Total worm biomass increased linearly with increasing numbers of worms. However, whilst the length and weight of male worms declined with increasing worm burden (8.4 and 24.6% respectively), female worms were less affected, only length showing a significant reduction with increasing parasite burden (16.0%). Therefore, increasing worm burdens impeded growth of P. muricola, but reduction in length and weight were relatively small in relation to the overall size of this nematode. Increasing worm burdens were associated with loss of host weight and reduction in stomach weight and worm burdens in excess of 20 exerted a measurable cost to the host, which in the field, may be associated with loss of overall host fitness.     

Trichinella spiralis: role of different life cycle phases in induction, maintenance, and expression of rapid expulsion in rats.

The capacity of different phases of the life cycle of Trichinella spiralis to induce rapid expulsion was examined. The phases examined included enteral preadults, enteral adults, and parenteral larvae. All had the ability to induce rapid expulsion although there were significant quantitative differences in their inductive capacity and in the kinetics of expression. Immunization with preadults required only a 48-hr enteral exposure to 2000 worms to induce strong rapid expulsion. In contrast rats required a 14-day exposure to adult worms to elicit a comparable response. After immunization with adults the reaction was demonstrable for only 2 weeks. Parenteral larvae produced only a weak rapid expulsion reaction by themselves and this response did not develop until some 8 weeks after challenge. When immunization with the enteral phases (preadult and adult) was combined with exposure to parenteral larvae a strong and enduring rapid expulsion reaction was observed. Phase specificity was also observed in the susceptibility of worms to the rapid expulsion response. The preadult phases, from infectious larvae to worms of up to 2 days of age were highly susceptible. Older worms, from 3 to 4 days old were not susceptible to rapid expulsion and could invade and establish themselves in the primed intestine for at least a 48-hr period without apparent adverse effects.     

Effect of host feeding and available glucose on glycogen synthase and phosphorylase activities in Hymenolepis diminuta and Vampirolepis microstoma

The influences of host feeding and the availability of glucose in vitro on the activities of glycogen synthase and glycogen phosphorylase in Hymenolepis diminuta and in Vampirolepis microstoma were studied. The worms were recovered from hosts that had been fed ad libitum, starved for 24 hr, or starved 24 hr and then refed for 1 hr immediately prior to worm recovery. The ratios of active to inactive glycogen synthase and phosphorylase were correlated with the host feeding regimen prior to recovery. Glycogen synthase in H. diminuta was predominately in the inactive D form in worms from both fed and fasted hosts. One hour after refeeding, up to 80% of the synthase was in the active I form. Phosphorylase in H. diminuta was predominantly in the active a form in worms from fed and fasted hosts, but activity of this enzyme was suppressed in worms from refed hosts. When H. diminuta from fasted hosts was incubated in a balanced salt solution containing 40 mM glucose, glycogen synthase I increased, and phosphorylase a decreased. Glycogen synthase in V. microstoma was predominantly in the inactive D form in worms from both the fed and fasted hosts, but the proportion in the active I form increased to over half the total synthase by 1 hr of host refeeding. The proportion of glycogen phosphorylase a was high in worms from fed hosts and decreased, but not dramatically, in worms from fasted hosts. The results suggested that the worms had access to another source of glucose, probably from the host bile, and we measured a low but significant concentration of carbohydrate in the gall bladder bile of mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis: quantitative relationships between intestinal worm burden, worm rejection, and the measurement of intestinal immunity in inbred mice

The effect of widely different doses of Trichinella spiralis muscle larvae on time to rejection of intestinal adults and on host survival was assessed in mice of the three rejection phenotypes; strong, intermediate, and weak. Rejection is weak with doses of less than 50 larvae per mouse. At these doses all mice rejected worms at a similar rate and no phenotypic variation was evident among strains. In contrast, rejection time was shortest for all strains and phenotypic variation among strains was evident in the range 50-100 muscle larvae/mouse. Above this dose the time taken to rejection increases monotonically with dose for all mouse strains examined. Despite this, the relative strength of rejection (i.e., phenotype) of a given strain of mouse was not changed at higher doses. Based on an end point of 98% rejection of the infective dose, time to rejection was predictable to +/- 1 day for all mouse strains and doses tested over the range 100-1000 worms administered. The principal reason for the increased time to complete rejection with larger worm doses was a delay in the initiation of intestinal rejection. This delay was evident above a dose of 50-100 larvae per mouse and occurred in all strains. Once begun, rejection was faster and eliminated more worms in unit time at higher doses (400-800 more) than at lower doses of worms. This appeared to be due to a stronger immune response of the host at higher doses. However, the increase in the rate of rejection was still not as great as the increase in the dose. We postulate that the delay in rejection with increased dose is due to a requirement for a "critical mass" of effectors/worm required to cause rejection. As dose increases, more time is required to reach the level at which worm rejection commences. Deaths due to higher doses of worms also occurred in a strain-specific manner and were temporally biphasic. The intestinal phase of infection produced mortality from 1 to 5 days after infection and the strongest rejection phenotype (NFS) was also the most resistant to intestinal deaths. Deaths occurring after Day 5 were due to the parenterally migrating newborn larvae. The weakest rejection phenotype, that of the B10 congenics, was also the least resistant to intestinal deaths. An experimental formula describing 98% worm rejection time with different doses was derived from the data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental studies on the migration of Anisakis sp. larvae (Nematoda: ascaridida) into the flesh of herring, Clupea harengus L.

In two experiments, a batch of 38 herring from Shetland was gutted immediately on capture (0 h) and further batches were gutted 14 and 37 h after capture and storage on ice. The numbers of Anisakis larvae in the body-cavity and in the flesh (detected by pepsin-HCl digest) were then counted. The proportion of fish with worms in the flesh was higher at 14 and 37 h than at 0 h. The percentage of the total worm burden in the flesh increased between cash each time interval, which suggests that a large-scale migration of larvae into the flesh occurs in ungutted fish. There was a positive association between the numbers of larvae in the viscera and the numbers in the flesh. Most larvae occurred in the hypaxial muscles. The results are discussed in relation to the observations of earlier workers.     

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.     

Efficacy of mebendazole and albendazole against Trichinella spiralis in mice

Changes in the sensitivity of Trichinella spiralis to anthelmintic treatment during the first 3 days of infection in mice were studied. Oral administration of either mebendazole or albendazole at 6.25 mh/kg 2 hr after exposure to infection eliminated 95-100% of the worms as determined at necropsy on day 7 postinoculation. Beyond the first day of infection the sensitivity of the parasite to benzimidazole therapy was much reduced and an oral dose of 50 mg/kg was only partially but significantly active against the adult worms. Despite decline in drug sensitivity during the enteral phase, gavage administration of either mebendazole or albendazole at 50 mg/kg for 5 consecutive days during the invasive phase of infection significantly reduced (96 and 67%, respectively) the number of larvae subsequently recovered from host musculature on day 45 postinoculation.     

Adult worms of the rodent intestinal nematode,Strongyloides venezuelensis,successfully invade chick intestinal mucosa

In order to study the mucosal invasion of a rodent intestinal nematode in bird intestine, chicks were infected with the intestinal nematode of rodents, Strongyloides venezuelensis, by subcutaneous larva inoculation and adult worm implantation. No evidence was obtained for larvae reaching the lungs or the intestine after infective larva inoculation. Adult worms implanted in the small intestine invaded the mucosa and remained there at least for 24 h, whereas those implanted in the caecum were trapped by mucus, and did not invade the mucosa. Mucosal invasion of adult worms in the small intestine was confirmed by histological examination. The number of adult worms in the intestinal mucosal tissue dropped rapidly within the first 24 h, which was associated with infiltrating granulocytes around the worms. The present study suggests that S. venezuelensis adult worms are able to invade the intestinal tissue of chicks, which do not belong to the vertebrate class of its normal definitive host, but that they are eliminated rapidly by mucosal defense system of the bird.     

Intraepithelial infiltration of eosinophils and their contribution to the elimination of adult intestinal nematode,Strongyloides venezuelensis in mice

Eosinophils were examined for the capacity of attacking Strongyloides venezuelensis adult worms in the intestinal mucosa by using interleukin (IL)-5 transgenic mice. In IL-5 transgenic mice, most of the subcutaneously inoculated infective larvae were killed during migration, and only a few worms could reach the small intestine. When the same number of adult worms were surgically implanted in the small intestine of IL-5 transgenic and control mice, fecal egg output as well as the number of adult worms recovered from the intestine was significantly lower in IL-5 transgenic mice. In the intestinal mucosa of IL-5 transgenic mice, large number of eosinophils was present in the lamina propria even before adult worm implantation. The number of eosinophils increased significantly as early as 24 h after implantation and tripled by day 3, whereas mucosal eosinophilia remained low in wild-type mice. Most notably, eosinophils infiltrated into the intestinal epithelium and surrounded adult worms in IL-5 transgenic mice, which was never seen in wild-type control mice. However, IL-5 transgenic mice required the same period as normal mice to completely expel implanted adult worms. The amount of specific IgA as well as total IgA in the stool was high in IL-5 transgenic mice before adult worm implantation, and dropped rapidly after adult worm implantation. The present study suggests that eosinophils are capable of attacking adult nematodes in the intestinal epithelia, probably in conjunction with secretory IgA, although they are not enough for the complete worm expulsion.     

Size of inoculum dose regulates in part worm burdens, fecundity, and lengths in ovine Haemonchus contortus infections

Density-dependent factors frequently have been shown to regulate population parameters of free-living and parasitic helminths. To test the effects of various infection levels of Haemonchus contortus on fecundity and worm size, lambs were inoculated with 3,000, 10,000, and 30,000 infective larvae. Daily eggs per gram (epg) and daily total fecal production per lamb were monitored continuously. Worms were collected from abomasa on 6, 15, 22, and 30 days postinfection (PI). Female worms were smaller on each day in the high-dose group when compared to female worms from the low-dose group; males in the high-dose group were smaller from days 15 through 30 PI. The high- and medium-dose groups had higher mortality rates on day 30 PI, and fecundity (eggs/female/day) was 78% lower. Daily epg and daily total eggs/lamb/day were lower in the high-dose group. Fecundity and worm size were correlated with the log-transformed dose level but not with adult worm number. Early parasite and/or host responses apparently exert long-term negative effects on growth and reproduction relative to the size of the establishing population of H. contortus.     

The ability of Aedes aegypti mosquitoes to survive and transmit infective larvae of Brugia pahangi over successive blood meals

The mortality of Aedes aegypti mosquitoes increased; immediately following a blood meal containing microfilariae of Brugia pahangi, when infective larvae began to migrate out of the flight muscles and when infective larvae were lost from the mosquitoes during a blood meal. When infective mosquitoes took a second blood meal 86.2% of the infective larvae escaped from their bodies. However, only 50.3% escaped when mosquitoes fed through a thin layer of cotton. Infective larvae in the abdomen of the mosquitoes stood the least chance of escaping from the insects. When infective mosquitoes were offered a third blood meal four days later, the proportion of infective larvae in the head and labium had risen from 56.6% in the control group to 66.0% and 69.4% in the two test groups. At this third feed 54.7% and 75.7% of the infective larvae were lost from mosquitoes with a low and medium pre-feeding worm burden respectively. This suggests that the escape of infective larvae from mosquitoes with only a few worms is less efficient than from mosquitoes with a medium worm burden.     

A mechanism for anti-asialo GM 1 antibody-induced anaphylactoid response in mice infected with Trichinella pseudospiralis

Intravenous injection of anti-asialo GM 1 antibody into mice infected with Trichinella pseudospiralis resulted in rapid acute illness or death accompanied by a dramatic rise in hematocrit values in these animals. The described antibody-induced changes were reversible by intravenous infusion of Hanks' balanced salt solution (HBSS). These effects were not seen in uninfected mice or in Trichinella spiralis-infected mice injected with anti-asialo GM 1 antibody. Viability of T. spiralis or T. pseudospiralis infective L1 larvae, both isolated worms and those housed in muscle, was unaffected by exposure to anti-asialo GM 1 antibody and complement. Infectivity of larvae of T. pseudospiralis decreased significantly following exposure to anti-asialo GM 1 antibody. Release of protein by T. pseudospiralis infective L1 larvae during incubation in the presence of anti-asialo GM 1 antibody was significantly greater than that released by worms incubated in normal rabbit serum or HBSS. Protein released by infective L1 larvae of T. pseudospiralis was identified as Trichinella excretory/secretory antigens by immunoblot. Intravenous injection of T. pseudospiralis excretory/secretory products resulted in anaphylaxis in T. pseudospiralis-infected mice but not in uninfected or T. spiralis-infected mice. Excretory/secretory product-induced anaphylactoid response also was reversible by the intravenous injection of HBSS or by injection of an antihistamine. Significantly higher levels of total IgE were observed in sera from mice infected with T. pseudospiralis compared to uninfected or T. spiralis-infected mice. Binding of anti-asialo GM 1 antibody to the surface of T. pseudospiralis muscle larvae induced release of excretory/secretory antigen by the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)