Clogmia albipunctata (Williston, 1893) midgut physiology: pH control and functional relationship with Lower Diptera (nematoceran) especially with hematophagous species.

Clogmia albipunctata (Williston, 1893) is a non-hematophagous insect belonging to the order Diptera, suborder Nematocera (Lower Diptera) and family Psychodidae. In the present work, we investigated how C. albipunctata control their midgut pH under different physiological conditions, comparing their midgut physiology with some nematoceran hematophagous species. The C. albipunctata midgut pH was measured after ingestion of sugar, protein and under the effect of the alkalinizing hormone released in the hemolymph of the hematophagous sand fly Lutzomyia longipalpis obtained just after a blood meal. The midgut pH of unfed or sugar-fed C. albipunctata is 5.5-6, and its midgut underwent alkalinization after protein ingestion or under treatment with hemolymph collected from blood fed L. longipalpis. These results suggested that in nematocerans, mechanisms for pH control seem shared between hematophagous and non-hematophagous species. This kind of pH control is convenient for successful blood digestion. The independent evolution of many hematophagous groups from the Lower Diptera suggests that characteristics involved in midgut pH control were already present in non-hematophagous species and represent a readiness for adaptation to this feeding mode.

Inhibition of vertebrate complement system by hematophagous arthropods: inhibitory molecules, mechanisms, physiological roles, and applications.

In arthropods, hematophagy has arisen several times throughout evolution. This specialized feeding behavior offered a highly nutritious diet obtained during blood feeds. On the other hand, blood-sucking arthropods must overcome problems brought on by blood intake and digestion. Host blood complement acts on the bite site and is still active after ingestion, so complement activation is a potential threat to the host's skin feeding environment and to the arthropod gut enterocytes. During evolution, blood-sucking arthropods have selected, either in their saliva or gut, anticomplement molecules that inactivate host blood complement. This review presents an overview of the complement system and discusses the arthropod's salivary and gut anticomplement molecules studied to date, exploring their mechanism of action and other aspects related to the arthropod-host-pathogen interface. The possible therapeutic applications of arthropod's anticomplement molecules are also discussed.

Evasion of the complement system by Leishmania through the uptake of C4bBP, a complement regulatory protein, and probably by the action of GP63 on C4b molecules deposited on parasite surface.

The complement system is a primary component of the vertebrate innate immune system, and its activity is harmful to microorganisms and parasites. To evade complement attack, some pathogens, such as viruses, bacteria, and protozoa, can interact with complement regulatory proteins from their hosts. Our research group has described the ability of Leishmania species to bind Factor H from human serum and use it as a tool to evade the complement system. However, there is no description of the interaction of Leishmania with other complement regulatory proteins, such as the C4b-binding protein (C4bBP), a negative regulator of classical and lectins complement system pathways. The results presented in this manuscript suggest that Leishmania infantum, L. amazonensis, and L. braziliensis recruit C4bBP from human serum. The uptake of C4bBP by L. infantum was studied in detail to improve our understanding of this inhibitory mechanism. When exposed to this complement regulator, parasites with inactivated GP63 bind to C4bBP and inactivate C4b deposited on their surface after serum exposure. This inactivation occurs by the action of Factor I, a complement system protease. In addition to the C4bBP-Factor I inactivation mechanism, the surface parasite protease GP63 can also inactivate soluble C4b molecules and probably that C4b molecules deposited on the parasites surface. This manuscript shows that Leishmania has two independent strategies to inactivate C4b molecules, preventing the progress of classical and lectins pathways. The identification of the C4bBP receptor on the Leishmania membrane may provide a new vaccine target to fight leishmaniasis.

Leishmania enriettii visceralises in the trachea, lungs, and spleen of Cavia porcellus.

BACKGROUND: Leishmania (Mundinia) enriettii is a species commonly found in the guinea pig, Cavia porcellus. Although it is a dermotropic species, there is still an uncertainty regarding its ability to visceralise during Leishmania life cycle. OBJECTIVE: Here, we investigated the ability of L. enriettii (strain L88) to visceralise in lungs, trachea, spleen, and liver of C. porcellus, its natural vertebrate host. METHODS: Animals were infected sub-cutaneously in the nose and followed for 12 weeks using histological (hematoxilin-eosin) and molecular tools (polymerase chain reaction-restriction fragment length polymorphism - PCR-RFLP). To isolate parasite from C. porcellus, animals were experimentally infected for viscera removal and PCR typing targeting hsp70 gene. FINDINGS: Histological analysis revealed intense and diffuse inflammation with the presence of amastigotes in the trachea, lung, and spleen up to 12 weeks post-infection (PI). Molecular analysis of paraffin-embedded tissues detected parasite DNA in the trachea and spleen between the 4th and 8th weeks PI. At the 12th PI, no parasite DNA was detected in any of the organs. To confirm that the spleen could serve as a temporary site for L. enriettii, we performed additional in vivo experiments. During 6th week PI, the parasite was isolated from the spleen confirming previous histopathological and PCR observations. MAIN CONCLUSION: Leishmania enriettii (strain L88) was able to visceralise in the trachea, lung, and spleen of C. porcellus.

How to get away with murder: The multiple strategies employed by pathogenic protozoa to avoid complement killing.

Parasitic protozoa are eukaryotic unicellular organisms that depend on a variety of living organisms and can develop intra- and extracellularly inside their hosts. In humans, these parasites cause diseases with a significant impact on public health, such as malaria, toxoplasmosis, Chagas disease, leishmaniasis and amebiasis. The ability of a parasite in establishing a successful infection depends on a series of intricate evolutionarily selected adaptations, which include the development of molecular and cellular strategies to evade the host immune system effector mechanisms. The complement system is one of the main effector mechanisms and the first humoral shield of hosts innate immunity against pathogens. For unicellular pathogens, such as protozoa, bacteria and fungi, the activation of the complement system may culminate in the elimination of the invader mainly via 1- the formation of a pore that depolarizes the plasma membrane of the parasite, causing cell lysis; 2- opsonization and killing by phagocytes; 3- increasing vascular permeability while also recruiting neutrophils to the site of activation. Numerous strategies to avoid complement activation have been reported for parasitic protozoa, such as 1- sequestration of complement system regulatory proteins produced by the host, 2- expression of complement system regulatory proteins, 3- proteolytic cleavage of different complement effector molecules, 4- formation of a physical glycolipid barrier that prevents deposition of complement molecules on the plasma membrane, and 5- removal, by endocytosis, of complement molecules bound to plasma membrane. In this review, we revisit the different strategies of blocking various stages of complement activation described for the main species of parasitic protozoa, present the most recent discoveries in the field and discuss new perspectives on yet neglected strategies and possible new evasion mechanisms.

cAMP: A second messenger involved in the mechanism of midgut alkalinization in Lutzomyia longipalpis.

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in the Americas. Female sand flies ingest sugar-rich solutions and blood, which are digested in the midgut. Digestion of nutrients is an essential function performed by digestive enzymes, which require appropriate physiological conditions. One of the main aspects that influence enzymatic activity is the gut pH, which must be tightly controlled. Considering second messengers are frequently involved in the coordination of tightly regulated physiological events, we investigated if the second messenger cAMP would participate in the process of alkalinization in the abdominal midgut of female L. longipalpis. In midguts containing the indicator dye bromothymol-blue, cAMP stimulated the alkalinization of the midgut lumen. Through another technique based on the use of fluorescein as a pH indicator, we propose that cAMP is involved in the alkalinization of the midgut by activating HCO3- transport from the enterocyte's cytoplasm to the lumen. The results strongly suggested that the carrier responsible for this process would be a HCO3- /Cl- antiporter located in the enterocytes' apical membrane. Hematophagy promotes the release of alkalinizing hormones in the hemolymph; however, when the enzyme adenylyl cyclase, responsible for cAMP production, was inhibited, we observed that the hemolymph from blood-fed L. longipalpis' females did not stimulate midgut alkalinization. This result indicated that hormone-stimulated alkalinization is mediated by cAMP. In the present study, we provide evidences that cAMP has a key role in the control of intestinal pH.

Leishmania enriettii visceralises in the trachea, lungs, and spleen of Cavia porcellus

BACKGROUND Leishmania (Mundinia) enriettii is a species commonly found in the guinea pig, Cavia porcellus. Although it is a dermotropic species, there is still an uncertainty regarding its ability to visceralise during Leishmania life cycle. OBJECTIVE Here, we investigated the ability of L. enriettii (strain L88) to visceralise in lungs, trachea, spleen, and liver of C. porcellus, its natural vertebrate host. METHODS Animals were infected sub-cutaneously in the nose and followed for 12 weeks using histological (hematoxilin-eosin) and molecular tools (polymerase chain reaction-restriction fragment length polymorphism - PCR-RFLP). To isolate parasite from C. porcellus, animals were experimentally infected for viscera removal and PCR typing targeting hsp70 gene. FINDINGS Histological analysis revealed intense and diffuse inflammation with the presence of amastigotes in the trachea, lung, and spleen up to 12 weeks post-infection (PI). Molecular analysis of paraffin-embedded tissues detected parasite DNA in the trachea and spleen between the 4th and 8th weeks PI. At the 12th PI, no parasite DNA was detected in any of the organs. To confirm that the spleen could serve as a temporary site for L. enriettii, we performed additional in vivo experiments. During 6th week PI, the parasite was isolated from the spleen confirming previous histopathological and PCR observations. MAIN CONCLUSION Leishmania enriettii (strain L88) was able to visceralise in the trachea, lung, and spleen of C. porcellus.

Evasion of the complement system by Leishmania through the uptake of factor H, a complement regulatory protein.

Escaping the complement system is an important step in the establishment of infections. Some pathogens have acquired the ability to inactivate the complement system to ensure successful infection. This has been observed in parasites from the genus Leishmania, which inactivate C3b molecules deposited on their surface through the membrane protease GP63. In the present study, we describe a new mechanism that also acts through C3b inactivation. This mechanism involves the binding of the complement regulatory molecule factor H from serum. Factor H signals a plasma protease (factor I) to inactivate C3b molecules deposited on the surface of the parasites. According to our results, Leishmania infantum, L. amazonensis, and L. braziliensis recruit factor H from human serum. The absorption of factor H by L. infantum was studied in detail to better understand how it works. L. infantum binds factor H from human serum and factor H-like proteins from dog serum. When exposed to purified factor H, promastigotes bind this regulatory molecule and inactivate C3b in the presence of factor I. This indicates the existence of an as yet unidentified factor H-binding outer surface molecule functioning as a receptor. The two mechanisms (GP63 and factor H binding) work independently, as Leishmania promastigotes with inhibited GP63 can easily inactivate C3b molecules on the surface of the parasite. The identification of the factor H receptor could lead to the development of a vaccine target for leishmaniasis control, as blocking antibodies to factor H binding could impair the mechanism of C3b inactivation, making the parasite more susceptible to the complement system.

Bedbug salivation patterns during hematophagy in the skin of a mammalian host.

Cimex lectularius (Hemiptera:Cimicidae) infestations have increased over the past decades in several parts of the world, constituting a major urban pest with no reversion signs. The impact on human health caused by these insects, commonly known as bedbugs, is associated with their obligatory hematophagous habit. Allergies induced by hematophagous arthropod bites are related to the deposition of salivary molecules in the host tissues. Many reports of humans developing severe allergic reactions due to bedbug bites have been recorded, however, there is limited information on the salivation of bedbugs on the host, which was the objective of this study. C. lectularius females were fed on blood containing acridine orange fluorochrome, which labeled the principal salivary glands content. The salivation pattern of bedbugs was investigated using intravital microscopy during its blood meal on the ear skin of hairless mice. Saliva deposition occurred during all insect blood-feeding phases, beginning as soon as the mouthpart touched the host skin. During the probing phase, saliva was deposited in large quantities in the host dermis. In contrast, during the engorgement phase (which represents the largest blood meal of the insects), saliva was released at a much slower rate. The apparent release of saliva into the cannulated vessel and/or adjacent tissue occurs only sporadically during insect blood ingestion. However, a small area (spot) of fluorescence was detected around the proboscis tip during this feeding phase. An interesting feature of bedbugs is that they release saliva inside and outside the vessels without removing their mouthparts from the vessel lumen. This is an effective feeding strategy because it does not interrupt blood ingestion and decreases the mouthparts movements on the host's skin, minimizing the damage to tissues and contact time with the host (feeding time).

The role of LuloPAT amino acid/proton symporters in midgut alkalinization in the sandfly Lutzomyia longipalpis (Diptera - Psychodidae).

In Lutzomyia longipalpis females, which are the main vectors of Leishmania infantum in the Americas, hematophagy is crucial for ovary development. The control of pH in the midgut during blood digestion is important to the functioning of the digestive enzymes, which release amino acids in the luminal compartment that are then transported through the enterocytes to the hemolymph for delivery to the ovary and other organs. In the present work, we investigated transport systems known as LuloPATs that are present in the midgut of L. longipalpis but not in other organs. These transporters achieve symport of amino acids with H+ ions, and one of them (LuloPAT1) is orthologous to a transporter described in Aedes aegypti. According to our results, the transcription levels of LuloPAT1 increased significantly immediately after a blood meal. Based on the variation of the fluorescence of fluorescein with the pH of the medium, we developed a technique that shows the acidification of the cytoplasm of gut cells when amino acids are cotransported with H+ from the lumen into the enterocytes. In our experiments, the midguts of the sandflies were dissected and opened longitudinally so that added amino acids could enter the enterocytes via the lumen (PAT carriers are apical). LuloPAT1 transporters are part of a complex of mechanisms that act synergistically to promote gut alkalinization as soon as blood intake by the vector occurs. In dissected but not longitudinally opened midguts, added amino acids could only enter through the basolateral region of enterocytes. However, alkalinization of the lumen was observed because the entry of some amino acids into the cytoplasm of enterocytes triggers a luminal alkalinization mechanism independent of LuloPATs. These findings provide new perspectives that will enable the characterization of the set of signaling pathways involved in pH regulation within the L. longipalpis midgut.

How Lutzomyia longipalpis deals with the complement system present in the ingested blood: The role of soluble inhibitors and the adsorption of factor H by midgut.

Complement inhibitors are present in all hematophagous arthropods. Lutzomyia longipalpis is an important vector of Leishmania infantum, the etiologic agent of visceral leishmaniasis in the Americas. Studies with this vector identified complement inhibitors and respective inhibitory mechanisms. Despite the studies conducted with L. longipalpis, there is a gap in the knowledge about what happens in vivo with the complement present in the blood ingested. The experiments reported here show that the soluble inhibitor present in the intestinal lumen can act on the classical pathway of the human complement system by inhibiting the cascade soon after the activation of the C4 component. This means that this inhibitor can inhibit both the classical and lectin pathways. In the absence of salivary or gut inhibitors, the intestinal epithelium can activate the alternative pathway. At the same time, it can activate the lectin and the classical pathways by binding of MBL as well as by an antibody-independent C1 deposition mechanism. Without the salivary and intestinal inhibitors, the sand fly midgut epithelium may be more susceptible to complement attack as indicated by the C9/C3 deposition ratio when compared with intestines after a blood feed on a human host. In L. longipalpis, most of the C3 molecules present inside the midgut after a blood meal are found in their native form (not activated C3) or are present as iC3b (its inactivated form). C3b inactivation to iC3b, on the intestinal surface, is probably performed by a mechanism involving the uptake of factor H by the intestinal epithelium. Factor H is a negative complement regulator present in the plasma. Collectively, these results indicate how the complement inhibitors are necessary for a successful hematophagy in a sand fly model.

Inhibition of the complement system by saliva of Anopheles (Nyssorhynchus) aquasalis.

Anopheline mosquitoes are vectors of malaria parasites. Their saliva contains anti-hemostatic and immune-modulator molecules that favor blood feeding and parasite transmission. In this study, we describe the inhibition of the alternative pathway of the complement system (AP) by Anopheles aquasalis salivary gland extracts (SGE). According to our results, the inhibitor present in SGE acts on the initial step of the AP blocking deposition of C3b on the activation surfaces. Properdin, which is a positive regulatory molecule of the AP, binds to SGE. When SGE was treated with an excess of properdin, it was unable to inhibit the AP. Through SDS-PAGE analysis, A. aquasalis presented a salivary protein with the same molecular weight as recombinant complement inhibitors belonging to the SG7 family described in the saliva of other anopheline species. At least some SG7 proteins bind to properdin and are AP inhibitors. Searching for SG7 proteins in the A. aquasalis genome, we retrieved a salivary protein that shared an 85% identity with albicin, which is the salivary alternative pathway inhibitor from A. albimanus. This A. aquasalis sequence was also very similar (81% ID) to the SG7 protein from A. darlingi, which is also an AP inhibitor. Our results suggest that the salivary complement inhibitor from A. aquasalis is an SG7 protein that can inhibit the AP by binding to properdin and abrogating its stabilizing activity. Albicin, which is the SG7 from A. albimanus, can directly inhibit AP convertase. Given the high similarity of SG7 proteins, the SG7 from A. aquasalis may also directly inhibit AP convertase in the absence of properdin.

pH control in the midgut of Aedesaegypti under different nutritional conditions.

Aedes aegypti is one of the most important disease vectors in the world. Because their gut is the first site of interaction with pathogens, it is important to understand A. aegypti gut physiology. In this study, we investigated the mechanisms of pH control in the midgut of A. aegypti females under different nutritional conditions. We found that unfed females have an acidic midgut (pH âˆ¼6). The midgut of unfed insects is actively maintained at pH 6 regardless of the ingestion of either alkaline or acidic buffered solutions. V-ATPases are responsible for acidification after ingestion of alkaline solutions. In blood-fed females, the abdominal midgut becomes alkaline (pH 7.54), and the luminal pH decreases slightly throughout blood digestion. Only ingested proteins were able to trigger this abrupt increase in abdominal pH. The ingestion of amino acids, even at high concentrations, did not induce alkalinisation. During blood digestion, the thoracic midgut remains acidic, becoming a suitable compartment for carbohydrate digestion, which is in accordance with the higher alpha-glucolytic activity detected in this compartment. Ingestion of blood releases alkalising hormones in the haemolymph, which induce alkalinisation in ex vivo preparations. This study shows that adult A. aegypti females have a very similar gut physiology to that previously described for Lutzomyia longipalpis It is likely that all haematophagous Nematocera exhibit the same type of physiological behaviour.

Triatomines (Hemiptera, Reduviidae) blood intake: Physical constraints and biological adaptations.

In order to efficiently obtain blood from their vertebrate hosts, bloodsucking arthropods have undergone an evolutionary selection process leading to specialist adaptations in their feeding apparatus (mouthparts and suction pumps) and salivary molecules. These adaptations act to counteract haemostasis, inflammation, and immune responses in their vertebrate hosts. The association of haematophagous arthropods with vertebrate hosts during a blood feed allows the transmission of pathogens between their hosts and vectors in a tripartite interaction. Feeding mechanisms in haematophagous arthropod species have been the subject of studies over at least eight decades worldwide, as a consequence of the importance of vector-borne diseases and their impact on human health. Here we review studies of the feeding mechanisms of triatomine bugs, with a particular focus on factors that influence their feeding performance when obtaining a blood meal from different vertebrate hosts.

Physiological characterization of the hematophagy of Ornithodoros rostratus (Acari: Argasidae) on live hosts.

Ornithodoros rostratus is an argasid tick and its importance is based on its hematophagy and the resulting transmission of pathogens such as Rickettsia rickettsii and Coxiella burnetii to its vertebrate hosts. In the face of a lack of physiological studies related to hematophagy in argasid ticks, this paper aims to identify and characterize the events that occur throughout the feeding by O. rostratus on live hosts. Electrical signals and alterations on the feeding site were monitored using intravital microscopy and electromyography. The analyses allowed for the characterization of four distinct events: suction, salivation, chelicerae movements and inactivity. Feeding was divided into two distinct phases: (1) penetration of mouthparts (when only salivation and chelicerae movements occurred) and the formation of the feeding pool (salivation and chelicerae movements with the first signs of suction) and (2) engorgement, during which chelicerae movements ceased and blood intake took place in feeding complexes (salivation followed by suction). Variations in patterns of the electrical signals, suction frequency and salivation showed four distinct sub-phases: (2a) suction with electrical signals of irregular shape, increased suction frequency and decreased salivation frequency throughout blood feeding; (2b) suction with electrical signals of symmetrical shape, high suction rates (3.8 Hz on average) and feeding complexes lasting for 7.7 s; (2c) suction with electrical signals of irregular shape, high suction frequency and feeding complex lasting 11.5 s; and (2d) electrical signals with no profile and the longest feeding complexes (14.5 s). Blood feeding ended with the withdrawal of the mouthparts from the host's skin.

Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge.

Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the "LbSapSal" vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with "LbSapSal" is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-ß), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after "LbSapSal" immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the "LbSapSal" vaccination is a potential tool to control the Leishmania chagasi infection.

Life cycle of Ornithodoros rostratus (Acari: Argasidae) ticks feeding on mice under laboratory conditions.

Ornithodoros rostratus Aragão is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil. Only limited studies about O. rostratus have been conducted and several aspects of their life cycle differ among studies or remain unexplored. In order to better elucidate the biology of O. rostratus, the present work describes its life cycle when feeding on mice under laboratory conditions. To complete their life cycle on mice, O. rostratus goes through a larval stage, 3-6 nymphal instars (nymph 1-6) and adult male and female. Adults can be originated from nymph 3-6. Nymphs 4 with higher weight after feeding tend to originate adults. Adults originated from early instars tended to be lighter. Females tended to be heavier than males. Larvae needed on average 2.7 days to complete their blood meal whereas other instars ranged from 17.3 to 78.3 min. The capacity to ingest blood was higher in larvae and females in comparison to males. The preecdysis period ranged from 5 to 12.5 days. After one blood meal, females remain on average 15.2 ± 5.8 days laying 276.8 ± 137.2.9 eggs. Females originated from nymph 4 had similar oviposition time, egg incubation and conversion ingested blood/number of eggs produced, but presented lower initial weigh and weigh gain, generating fewer eggs. Our results added novel information on O. rostratus biology and was discussed considering the variability of argasid populations and in context with the differences about their life cycle described in previous works.

Host modulation by a parasite: how Leishmania infantum modifies the intestinal environment of Lutzomyia longipalpis to favor its development.

Some reports have described the interference of Leishmania on sand flies physiology, and such behavior most likely evolved to favor the development and transmission of the parasite. Most of these studies showed that Leishmania could modulate the level of proteases in the midgut after an infective blood meal, and decreased proteolytic activity is indeed beneficial for the development of promastigotes in the gut of sand flies. In the present study, we performed a detailed investigation of the intestinal pH in Lutzomyia longipalpis females naturally infected with Leishmania infantum and investigated the production of trypsin by these insects using different approaches. Our results allowed us to propose a mechanism by which these parasites interfere with the physiology of L. longipalpis to decrease the production of proteolytic enzymes. According to our hypothesis L. infantum promastigotes indirectly interfere with the production of trypsin by modulating the mechanism that controls the intestinal pH via the action of a yet non-identified substance released by promastigote forms inside the midgut. This substance is not an acid, whose action would be restrict on to release H+ to the medium, but is a substance that is able to interfere with midgut physiology through a mechanism involving pH control. According to our hypothesis, as the pH decreases, the proteolytic enzymes efficiency is also reduced, leading to a decline in the supply of amino acids to the enterocytes: this decline reduces the stimulus for protease production because it is regulated by the supply of amino acids, thus leading to a delay in digestion.

LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4⁺ and CD8⁺) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis.

BACKGROUND: The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8⁺ T cells, and high NO production. METHODS: We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 107 late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge. RESULTS: The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5⁺, CD4⁺, and CD8⁺ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load. CONCLUSIONS: These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.

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

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.