Transplantation of adult Trichinella spiralis between hosts: worm survival and immunological characteristics of the host--parasite relationship.

A technique for the transplantation of Trichinella spiralis worms directly into the host intestine is described. Infections established by the direct transfer of adult worms were essentially normal both in terms of their survival and reproduction and in their stimulation of, and susceptibility to, host immune responses. Worms transplanted from NIH mouse donors at intervals after infection had an equal ability to survive in the recipient, even when taken from the donor shortly before or during the process of worm expulsion, showing that expulsion does not require worms to be irreversibly damaged. It was noted, however, that after 7 days in the donor the ability of the worm to reproduce in the recipient was temporarily impaired.     

Nippostrongylus brasiliensis: further properties of antibody-damaged worms and induction of comparable damage by maintaining worms in vitro.

When adult Nippostrongylus brasiliensis were maintained in vitro they became damaged. Using the criteria of ultrastructural morphology, acetylcholinesterase isoenzyme pattern and the behaviour of the worms after transfer to a normal rat, this damage appeared to be similar to that produced by the in vivo action of antibodies. Antibodies were shown to be responsible for the anterior migration of adult worms which occurs during primary infections in mature rats and in the prolonged infections seen in lactating and immature rats. Antibody damaged worms and worms unaffected by antibodies were equally able to stimulate the immune response required for worm expulsion. Apparently antibody damage is not required for the initiation of the second immune component necessary for expulsion of this parasite.     

Synergistic interaction between immune serum and thoracic duct cells in the adoptive transfer of rapid expulsion of Trichinella spiralis in adult rats

Rapid expulsion of Trichinella spiralis could be transferred to naive adult rats with thoracic duct lymphocytes and immune serum. Thoracic duct cells collected from Days 3-5 and immune serum collected on Day 28, respectively, after infection were effective. Both cells and serum were unable to transfer rapid expulsion when given alone, even in large volumes. Recipients of immune serum and cells eliminated a significantly higher number of larvae than control rats by 1 hr after challenge with muscle larvae. Rapid expulsion produced 30-80% larval worm rejection but could not be increased by the transfer of more cells or immune serum. Mucus trappings did not appear to play a role in the rejection process. After transfer of 2 x 10(8) cells and 4.0 ml immune serum, rapid expulsion persisted for less than 1 week. However, after adoptive transfer of cells alone, the gut remained functionally receptive to the passive transfer of immune serum for 7 weeks. Therefore, the changes effected by transfer of cells were long lived in contrast to the 1 week, or less, of functional persistence by transferred immune serum. The data indicate that two separate processes, one cell mediated and the other immune serum mediated, interact synergistically in the intestine and lead to the expression of rapid expulsion.     

Strongyloides ratti: reversibility of immune damage to adult worms

Transplantation experiments were conducted to assess the reversibility or irreversibility of the damage sustained by Strongyloides ratti during infections in the rat host. Worms of different ages from primary and secondary infections were recovered from their original hosts and transplanted surgically into naive rats. The size and fecundity of normal (Days 6–11 postinfection) worms were maintained after transfer. Damaged worms from primary infection (Days 22–26) showed complete recovery of size and fecundity within 10 days of transfer; damaged worms from a secondary infection (Days 6–7) also showed functional recovery but to a lesser extent. The ultrastructural changes observed mainly in the intestine of damaged worms from primary infections, prior to their transfer, were, however, only partially ameliorated following transplantation into new naive hosts; there was no complete return to structural normality. On the other hand, second infection worms did show almost complete ultrastructural recovery. The course of a transplanted infection established with either damaged or normal worms was similar to infections established percutaneously. Increase in the size of transplanted infections from 100 to 250 worms per recipient did not alter the dynamics of the host/parasite relationship. There was no evidence of adaptation in S. ratti and damaged worms, when transplanted into naive rats, were as successful as normal worms in protecting the host against a subcutaneous larval infection. The implications of this work on the present understanding of the phenomenon of autoinfection in experimental rodent strongyloidiasis are discussed.     

Partial cross-resistance between Strongyloides venezuelensis and Nippostrongylus brasiliensis in rats.

Rats were immunized through an initial infection with 1,000 filariform larvae (L3) of Nippostrongylus brasiliensis and after complete expulsion of worms they were challenged with 1,000 L3 of Strongyloides venezuelensis to investigate whether cross-resistance developed against a heterologous parasite. Nippostrongylus brasiliensis-immunized rats developed a partial cross-resistance against S. venezuelensis migrating larvae (MSL3) in the lungs and adult worms in the small intestine. The population of MSL3 in the lungs were significantly lower (P < 0.05) in immunized rats (22.0 +/- 7.4) compared with controls (105.0 +/- 27.6). The populations of adult worms, egg output and fecundity were initially decreased but from day 14 post-challenge they did not show any significant difference between immunized and control rats. However, the length of worm in immunized rat was revealed as retardation. Peripheral blood eosinophilia was significantly decreased (P < 0.05) on day 7 post-challenge and then gradually increased, which peaked on day 42 post-challenge when most of the worms were expelled. These results suggest that peripheral blood eosinophilia is strongly involved in the worm establishment and expulsion mechanisms.     

Effects of PGE1 or PGE2 and/or acetazolamide on expulsion of Nippostrongylus brasiliensis from rats

PGE1 and PGE2 have been reported to enhance natural expulsion of Nippostrongylus brasiliensis, a nematode parasite, from the intestine of the rat. Mucus production may also be a key element of worm rejection. Our study attempts to determine if 1) PGE1 or PGE2 alter the normal course of infection with N. brasiliensis in rats, 2) a known mucous enhancing drug, acetazolamide, can augment the rate of worm expulsion, and 3) combinations of prostaglandins and acetazolamide affect N. brasiliensis in the rat. Rats were inoculated with approximately 1,000 infective larvae of N. brasiliensis. Animals were administered, intraduodenally, one of the following: 0.2 ml 0.9% NaCl; 0.2 ml 100% ethanol; 250 micrograms PGE1/0.2 ml 100% ethanol; 250 micrograms PGE2/0.2 ml 100% ethanol; 250 micrograms acetazolamide/0.2 ml 100% ethanol; 250 micrograms PGE1 or PGE2 + 250 micrograms acetazolamide/0.2 ml 100% ethanol. These solutions were given in a single bolus on day 6 postinoculation (PI) or twice daily on days 6-9 PI. Following these treatments the number of parasite ova per gram feces per day for days 6-10 PI and numbers of worms present at necropsy on day 10 PI were determined. Treatment with prostaglandins or acetazolamide or both failed to adversely affect egg deposition by adult female worms or the number of worms in the small intestine. These results do not support the involvement of prostaglandins in the expulsion of N. brasiliensis from the host intestine.     

Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat).

The biomass of 8-day-old worms of Hymenolepis diminuta in secondary infections, administered to rats 3-10 days after chemotherapeutically expelling a primary infection, was 70-90% less, and the worms were more posteriorly distributed, than in naive controls. The strong depressive effect on growth waned rapidly over 2-5 weeks, but even in rats not challenged until 17 months later, worm growth was weakly depressed by 30%. The extent to which growth was depressed in a secondary infection was independent of the number of worms in the challenge but increased with number of worms in the immunizing infection up to four to eight worms. Further increase up to 64 worms had little effect. This suggests, as it is known that the biomass of worms in a rat reaches a maximum with infections of between five and 10 worms, that the change in the intestine is proportional to biomass, not number, of worms. It is argued that partially suppressed immuno-inflammatory changes in the intestine, which will affect secondary worms so strongly, will also have depressed growth and fecundity effects on the primary worms, that a dynamic equilibrium is reached between the strength of the intestinal response and the biomass of the tapeworm, and that it is reaching this equilibrium, not a 'crowding effect', which limits H. diminuta to a level compatible with the survival of the rat.     

Angiostrongylus cantonensis: Rejection of pulmonary arterial transfers of lung stage worms

Experimental transfer of the lung stage worms of Angiostrongylus cantonensis was performed between permissive hosts (rats) and between permissive (rat) and nonpermissive hosts (guinea pigs and rabbits). These worms from rats were rejected when implanted into nonpermissive hosts. Unexpectedly, similar worms did not survive well even in permissive hosts; the majority of recipient rats did not have first-stage larvae (L₁) in their stools and, even when positive for L₁, the number of the larvae shed was few. These findings contrast with the successful pulmonary arterial transfer of younger, intracranial-stage worms. It was shown that differences in rat strain between donor and recipient had no significant effect on the subsequent worm survival in recipient hosts. The alteration of maintaining conditions of the intrapulmonary worms, prior to transfer, in terms of temperature, media, and maintaining period, also showed no profound effect on the subsequent worm survival. The kinetics of precipitating and reaginic antibody levels in rats implanted with the intrapulmonary worms were analogous to those in rats with intracranial-stage worms. The findings indicate that some qualitative differences may exist between the worms obtained from two different sites.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

Villous atrophy and expulsion of intestinal Trichinella spiralis are mediated by T cells.

The development of villous atrophy and crypt hyperplasia in, and expulsion of nematodes from, the small intestine of the mouse during Trichinella infection is shown to be mediated by T cells. During Trichinella infection, worms initially localise in the anterior half of the small intestine. Their expulsion from here after 6–8 days follows the onset of villous atrophy and crypt hyperplasia in the jejunum and the normal jejunal morphology is restored after complete expulsion of worms from the small intestine at 12–15 days. In thymectomised mice, according to the extent of T-cell depletion, worm localisation is atypical, expulsion is either delayed or absent, and villous atrophy and crypt hyperplasia are either delayed and reduced or absent. The adoptive immunization of infected thymectomised mice with mesenteric lymph node cells (including primed T blasts) from infected donors completely restores the normal host response and enhances the onset of crypt hyperplasia. These findings are discussed in relation to T-cell traffic and delayed-type hypersensitivity in the gut.     

Action of diethylcarbamazine citrate on protective immunity in rats infected with Nippostrongylus brasiliensis

Rats made immune to Nippostrongylus brasiliensis and treated with diethylcarbamazine citrate (DEC) orally (250 mg/kg X 6) exhibited significant suppression of functional immunity. Similarly, administration of compound 48/80 (100 micrograms/rat i.p.) made the immune rats susceptible to challenge infection. Treatment of rats, with 22-day infection with compound 48/80, histamine (20 mg/rat, per os), or L-histidine (20 mg/rat, orally s.c.) did not accelerate worm expulsion. A massive complement-dependent adherence of peritoneal cells (1 X 10(8], isolated from immune DEC-treated and untreated rats, to infective larvae (L3) was observed. Likewise, heavy congregation of normal peritoneal cells to larvae was noticed when the cells were incubated with sera obtained from immune, DEC-treated or untreated rats. The rats receiving mesenteric lymph node cells (125 X 10(6) i.v.) or sera (0.5 ml or 1 ml X 3 i.p.), obtained from immune DEC-treated rats and challenged with infective larvae developed 50% more worms than those which received cells or serum from untreated immune donors. DEC appears to cause suppression of functional immunity and worm expulsion is not histamine mediated.     

Schistosoma mansoni: challenge attrition during the lung phase of migration in vaccinated and serum-protected rats

Serum harvested from Sprague-Dawley rats twice vaccinated with gamma-irradiated cercariae of Schistosoma mansoni is able to protect naive recipients against a challenge infection, but the challenge parasites are susceptible to immune elimination over a very short period of time. Thus, vaccinated rat serum protects recipients against a percutaneous cercarial challenge when transferred on Day +5 but not Day 0 and protects recipients against a tail vein challenge with 5-day-old lung worms when transferred on Days 0 or +1, but not Days +4 or +5. Rats challenged with lung worms via the tail vein and given serum on Day +3 exhibit approximately half the protection expressed by counterparts that received serum on Day 0. However, vaccinated rat serum does not protect naive recipients against a lung worm challenge introduced directly into the liver. These data indicate that immune elimination of challenge parasites in the vaccinated rat model is site-dependent rather than stage-dependent, and most probably occurs during the lung phase of parasite migration.     

Studies on the involvement of prostaglandins and their precursors in the rejection of Nippostrongylus brasiliensis from the rat

In vitro incubation of 6-day Nippo-strongylus brasiliensis in the presence of PGE₁ at 1000 ng/ml and PGE₂ at 500–10,000 ng/ml of medium did not affect worm motility nor in vivo survival of worms implanted into the small intestine of recipient rats. The intraduodenal injection of 250 and 500 μg PGE₁ or PGE₂ did not lead to expulsion of worms from infected rats. An in vitro exposure to precursor fatty acids of PGE₁ and PGE₂, dihomo-γ-linolenic acid and arachidonic acid, respectively, at concentrations of 1000–15,000 ng/ml of medium also failed to inhibit worm motility and in vivo worm survival. These results are at variance with some earlier reports and do not suggest that prostaglandins are directly involved in the immune rejection of N. brasiliensis. No prostaglandins could be demonstrated in worm homogenates.     

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.     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

Trichinella spiralis: immunity and inflammation in the expulsion of transplanted adult worms from mice.

The technique of implanting adult Trichinella spiralis into the intestines of mice has been used to assess the contributions of direct, anti-worm immunity and of intestinal inflammation to worm expulsion. The survival after transfer of worms exposed to an effective adoptive immunity in donors was no different from that of worms taken from control donors. Worms taken from donors 8 days after infection, i.e., shortly before the onset of expulsion, showed no increased susceptibility to an immunity adoptively transferred to the recipient mice. When worms were implanted into mice responding to a prior, oral infection they were expelled rapidly. This expulsion was independent of the age of the worms transferred and took place at the same time as the expulsion of the existing infection.     

Immunity to Trichinella spiralis in irradiated mice

Irradiation prevented the accelerated expulsion of Trichinella spiralis from mice immunized by transfer of immune mesenteric lymph node cells (IMLNC) or by prior infection. Nevertheless, worms in irradiated immune mice were smaller and less fecund than those in controls. In adoptively immunized and irradiated mice expulsion could not be achieved by increasing the numbers of IMLNC transferred, although the effect upon worm length was more severe. Thus IMLNC express a direct, anti-worm immunity which is independent of their role in worm expulsion. IMLNC cause expulsion in irradiated mice only when adequate levels of bone marrow-derived cells are available. The results are discussed in terms of a possible antibody-mediated basis for direct anti-worm immunity.     

Some influences of population density on Hymenolepis diminuta in rats.

When measured 56 days postinfection the length, wet weight and dry weight of Hymenolepis diminuta were all found to decrease with increasing number of cysticercoids given up to 20. The mean position of the worms in 10, 12 and 20 worm infections is significantly posterior to that of 1, 2 and 5 worm infections and the worms are attached over a wider area of the intestine. Egg production by the worms was followed up to day 56 postinfection; the number of eggs produced per worm and even per rat decreased with increasing population density. Thus the best way to get most eggs and to maintain the parasite in the laboratory is to have rats infected with only one tapeworm. Rats given 1-20 cysticercoids showed a mean recovery of 100-65%, while rats given 40-200 cysticercoids showed a mean recovery ranging from 13 to 2%. In addition to 'normal' worms, defined as worms greater than 10 mm, small, most probably destrobilated, worms were found. In the 50 and 100 cysticercoid infections, worm recoveries were, respectively, 8% 'normal', 16% small, and 2% 'normal', 5% small. From the significantly lower recovery from heavy infections it is concluded that a deleterious factor is operating during the 8 weeks after the infection.     

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.