Trypanosoma cruzi: metacyclogenesis in vitro--I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods.

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasites were maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuated virulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Biological properties of Trypanosoma cruzi metacyclic trypomastigotes: usefulness for studying Chagas' disease (brief report).

We have investigated changes in the biological properties of metacyclic trypomastigotes obtained from various sources, kept in the laboratory under diverse conditions and subjected to different procedures. Results demonstrate the great capacity of adaptation of Trypanosoma cruzi to changeable environments. The usefulness of different chemically defined media and consecutive passages through various hosts emphasize the importance of mimicking the life cycle of the parasite.

Experimental chemotherapy of Schistosoma mansoni with praziquantel and oxamniquine: differential effect of single or combined formulations of drugs on various strains and on both sexes of the parasite.

The susceptibility of two Venezuelan (YT and SM) and one Brazilian (BH) strain of Schistosoma mansoni to single oral doses of praziquantel (Pz; 250 or 500 mg/kg), oxamniquine (Ox; 40, 60, or 100 mg/kg) or to low-dose combinations of both drugs (33 mg/kg Pz and 25 mg/kg Ox; 66 mg/kg Pz and 12.5 mg/kg Ox; 250 mg/kg Pz and 40 mg/kg Ox) was experimentally evaluated in mice. At lower doses of either drug, adult worms of the SM isolate were less susceptible than those of the BH and YT isolates. However, no difference in liver or intestinal egg counts (IECs) could be detected among the isolates after this treatment. At such doses, Pz was better than Ox at reducing IECs. In spite of lowered IECs, eggs continued to accumulate in the liver after Ox treatment. At higher individual doses or following treatment with low-dose combinations of both drugs, no difference in susceptibility could be detected among the parasite isolates. Under such conditions, oviposition was drastically reduced in all three isolates. We confirm that Ox preferentially kills male parasites and present for the first time evidence for the preferential killing of female worms by Pz. We propose that the synergistic effect obtained in the present study and in other investigations using low-dose combinations of both drugs may be due to the preferential cytotoxicity of each drug against a different parasite sex.

Evidence that radio-sensitive cells are central to skin-phase protective immunity in CBA/Ca mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni as well as in naive mice protected with vaccine serum.

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were subjected to 550 rad of whole body (gamma) irradiation and then challenged 3 days later with normal cercariae. The perfusion recovery data showed that this procedure reduced the primary worm burden in naive mice by 22% and the challenge worm burden in vaccinated mice by 82%. Irradiation also ablated the peripheral blood leucocytes of both mouse groups by 90-100% at the time of challenge. Histological data revealed that such treatment caused a dramatic change in number, size and leucocyte composition of cutaneous inflammatory skin reactions that characterize challenged vaccinated mice and are known to entrap invading larvae; cutaneous eosinophils were preferentially abolished by this treatment. Polyvaccine mouse serum that conferred protection passively upon naive recipient mice, failed to protect naive/irradiated mice when administered by the same protocol. Distraction of macrophages by treatment of mice with silica did not affect the establishment of a primary worm burden and reduced the protection exhibited by vaccinated mice by only 16%. These data indicate that radio-sensitive cells are important to both innate and specific acquired resistance in this mouse model and that macrophages contribute only marginally to the expression of vaccine immunity.

Schistosoma mansoni: analysis of the humoral and cellular basis of resistance in guinea-pigs vaccinated with radiation-attenuated cercariae.

This study addresses the humoral and cellular basis of specific acquired immunity in the guinea-pig irradiated vaccine model of schistosomiasis mansoni. Rodents vaccinated with 500 gamma-irradiated cercariae and then splenectomized 4.5 weeks later showed a 33% reduction in resistance to challenge as compared to vaccinated animals or vaccinated/sham splenectomized controls. Serum harvested from once vaccinated individuals conferred modest levels of resistance upon naive recipients in some experiments, but transfer was not achieved consistently. Serum from vaccinated and thrice boosted rodents (Vbbb) routinely transferred around 45% immunity, however, provided it was given in 4 ml aliquots on day 9 post-challenge; Vbbb serum thus transferred 50% of donor immunity. Interestingly, multiple doses of this protective serum given on and either side of day 9 did not enhance the protection achieved with a single 4 ml aliquot. Neither peripheral lymph node cells nor splenocytes from the polyvaccinated serum donors were able to transfer resistance to recipient guinea-pigs and they failed to augment the protection achieved with Vbbb serum. Foot-pad testing revealed no correlation between delayed hypersensitivity responses and immunity to challenge in vaccinated guinea-pigs. Although polyvaccine guinea-pig serum successfully protected homologous recipients, it failed to protect mice when administered either at the time of challenge (the optimal schedule for transfer of polyvaccine mouse serum), or around day 9 (the optimal schedule for guinea-pigs). Similarly, guinea-pigs could not be protected with polyvaccine rat serum that conferred 75% resistance upon naive recipient rats.

Schistosoma mansoni: evidence that site-dependent host responses determine when and where vaccine immunity is expressed in different rodent species.

Laboratory rodents vaccinated with highly irradiated cercariae of Schistosoma mansoni develop significant levels of specific acquired resistance yet effect challenge elimination in different organs. Mice and guinea-pigs are at opposite ends of the spectrum in this respect since, in our hands, vaccinated mice kill challenge parasites in the skin whereas vaccinated guinea-pigs kill challenge parasites predominantly in the liver. To determine whether this phenomenon is host-dependent (site) or parasite-dependent (stage), we have transferred worms harvested from mice or guinea-pigs into vaccinated recipient guinea-pigs. The results show that mouse-derived 5-day lung worms and 9-day liver worms that are essentially refractory to vaccine resistance in mice are indeed susceptible to vaccine resistance in guinea-pigs. Identical levels of susceptibility were recorded for lung-stage larvae introduced via the foot vein so as to experience lung and liver mechanisms, or via the mesenteric vein to bypass the lung, thereby confirming that vaccine resistance in guinea-pigs operates in the liver. Mouse worms and guinea-pig worms exhibited equivalent levels of susceptibility at all stages of development. Thirteen-day-old larvae from either donor species were on the border-line of vulnerability, while 20-day-old worms were totally refractory to vaccine immunity in guinea-pigs. These data show that vaccine immunity in different rodent species is a site-dependent, rather than a stage-dependent phenomenon. There is, however, an upper age limit of schistosome vulnerability which is common to worms harvested from different donor species.

Variable generation of specific acquired resistance in CBA/Ca mice chronically infected with schistosomiasis mansoni.

Naive CBA/Ca mice and CBA/Ca mice infected 12 weeks previously with 20 normal cercariae of Schistosoma mansoni were challenged percutaneously with isotopically labelled parasites. Challenge worm migration was followed, in 5 separate experiments, by means of compressed organ autoradiography. In three experiments, 43.1% of challenge parasites did not arrive in the lungs of infected mice on day 6 as compared to 7.7% in naive controls, thereby indicating that pre-lung sites constitute the first barrier in resistance to reinfection. A further 15% of the challenge worm burden was lost in the lungs or en route to the liver in the immune animals, and portal perfusion revealed that 25.4% of the challenge was lost in the liver. Two other experiments revealed no comparable phases of pre-liver attrition however; instead resistance was only evident at final perfusion on days 28 or 35. These data reveal the variable generation of specific acquired immunity in mice harbouring a chronic schistosome infection and thus clarify current discrepancies in the literature. The results are discussed in relation to documented evidence for the nature of specific and non-specific immune mechanisms reported to operate at different sites in the infection model of schistosomiasis mansoni.

Schistosoma mansoni: enhanced efficacy of praziquantel treatment in immune mice.

Naive CBA/Ca mice and CBA/Ca mice vaccinated 4 weeks previously with radiation-attenuated cercariae of Schistosoma mansoni were challenged with normal cercariae and then treated with 500 mg/kg body weight of Praziquantel (Pzq). The drug was administered intramuscularly or intradermally on day 1, but intramuscularly only on day 6. The results show clearly that in naive mice skin-stage larvae were susceptible to Pzq provided the drug was given intradermally on day 1. Lung worms were susceptible to Pzq given intramuscularly on day 6. Drug efficacy in naive mice is thus dependent upon treatment being given at the correct time and via the optimal route. The efficiency of Pzq treatment was enhanced in vaccinated mice, but was again affected by the treatment regime. Analysis of the data revealed a highly significant synergistic effect between drug treatment and vaccination when Pzq was given intramuscularly on day 6. Synergy was detectable but only marginally significant when the drug was administered intradermally on day 1, and could not be demonstrated when Pzq was given via the intramuscular route on day 1. These findings are discussed in the light of known sites and mechanisms of vaccine resistance in mice, as well as in relation to the mode of action of Pzq against schistosome parasites.