Different host complement systems and their interactions with saliva from Lutzomyia longipalpis (Diptera, Psychodidae) and Leishmania infantum promastigotes

Abstract

Background: Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva.

Results: The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays.

Conclusion: Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

Figure 1. Influence of pH in the activation of complement system from different hosts.

Activation of classical (A) and alternative (B) pathways from the serum of dogs, guinea pigs, rats and chickens at pH 7.4 and 8.15.

Figure 2. Effect of L. longipalpis saliva on the classical pathway of the complement system from different hosts at pH 7.4 and pH 8.15.

Figure 3. Effect of L. longipalpis saliva on the complement alternative pathway of different hosts at pH 7.4 and pH 8.15.

The guinea pig alternative pathway could not be assessed at pH 8.15 because the alkalinisation itself affects the pathway's efficiency in a way that the difference between the positive and negative controls is undetectable. The results are expressed as the mean of percentage of haemolysis ±SD. Three replicates were performed for the experiments.

Figure 4. Effect of L. longipalpis intestinal content on the classical and alternative complement pathways of chickens at pHs of 7.4 and 8.15.

The results are expressed as the mean of percentage of haemolysis ±SD. Three replicates were performed for each experiment.

Figure 5. Effect of L. longipalpis saliva on the C3b deposition of classical and alternative complement pathways of dogs and guinea pigs at pHs of 7.4 and 8.15.

The guinea pig alternative pathway could not be assessed at a pH of 8.15 because the alkalinisation itself affects the pathway's efficiency in a way that the difference between the positive and negative controls is undetectable. The results are expressed as the mean of percentage of C3b deposition ± SD. Three replicates were performed per experiment.

Figure 6. Kinetic lysis of L. infantum promastigotes by chicken sera.

Effect of different concentrations of chicken sera (A) and different incubation temperatures (B) on the viability of L. Infantum promastigotes. The results are expressed as the mean of the percentage of live cells ± SD. Three replicates were performed for each experiment.

Figure 7. Kinetic lysis of L. infantum promastigotes by different concentrations of chicken sera in the presence and absence of L. longipalpis saliva.

The results are expressed as the mean of the percentage of live cells ± SD. Three replicates were performed for each experiment.