Nelder F. Gontijo

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity.

Abstract The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) GT (total anti-Leishmania IgG), IgG1 and IgG2 subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG2 concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.

Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis.

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(beta 1,4)Man(alpha 1)-PO(4) backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi. The distinguishing feature of LPG of procyclic L. chagasi consisted of beta 1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite-sand fly interactions.

Anti-complement activity in the saliva of phlebotomine sand flies and other haematophagous insects

The saliva of haematophagous insects has a series of pharmacological activities which may favour blood feeding. In the present study, an inhibitory effect on the complement system was observed in salivary extracts obtained from the phlebotomine sand flies Lutzomyia longipalpis and Lu. migonei. Saliva from Lu. longipalpis was capable of inhibiting both the classical and alternative pathways, while that from Lu. migonei acted only on the former. Other haematophagous insect species were screened for inhibition of the classical pathway. The triatomine bugs Panstrongylus megistus, Triatoma brasiliensis and Rhodnius prolixus were also able to inhibit the classical pathway whereas the mosquito Aedes aegyti and flea Ctenocephalides felis were not. The activity of Lu. longipalpis saliva on the classical pathway was partially characterized. The inhibitor is a protein of Mr 10000-30000 Da, which is very resistant to denaturation by heat. The inhibition of the complement system by phlebotomine sand flies may have a role in the transmission of Leishmania to the vertebrate hosts. The inhibitor molecule is thus a promising component of a vaccine to target salivary immunomodulators.

Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR

BackgroundRhodnius prolixus is a blood-feeding insect that can transmit Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and R. prolixus has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR.ResultsThe expression stability of five candidate reference genes (18S rRNA, GAPDH, β-actin, α-tubulin and ribosomal protein L26) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with T. cruzi or T. rangeli. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. 18S, GAPDH and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. β-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. L26 was identified as the poorest reference gene in the studies performed.ConclusionsThe expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in R. prolixus studies.

Feeding behaviour of morphologically similar Rhodnius species: influence of mechanical characteristics and salivary function.

Despite their morphological similarities, very similar Rhodnius species (R. prolixus, R. robustus, R. nasutus and R. neglectus) displayed a distinct feeding behaviour when fed on artificial feeder, pigeon or mouse. On pigeon hosts, these species showed distinct groups in terms of cumulative probing time - quicker species (R. prolixus and R. neglectus) followed by R. nasutus and finally a much slower species (R. robustus). On mouse hosts, R. nasutus showed quicker probing time compared to the other three species. Moreover, R. prolixus displayed quicker probing time compared to R. robustus and R. neglectus. Except for R. nasutus, the mean total ingestion rate tended to have different values between feeding sources (artificial feeder>pigeon>mouse). The volume ingested by each cibarial pump contraction and maximum frequency obtained using the artificial feeder are expected to be related to intrinsic mechanical characteristics of the insect feeding apparatus. However, probing time and the modulation of cibarial pump frequency on live hosts may be related to salivary function. R. prolixus showed high mechanical and salivary efficiency, achieving high values of total ingestion rate when fed on artificial feeder or either of the hosts. Comparative analysis suggests that species which possess higher total ingestion rates tend to achieve higher nutritional status, allowing them to reach higher densities.

Comparison of feeding behaviour of Triatoma infestans, Triatoma brasiliensis and Triatoma pseudomaculata in different hosts by electronic monitoring of the cibarial pump

Feeding behaviour of Triatoma infestans, T. brasiliensis and T. pseudomaculata on pigeons and mice was compared by electronic monitoring of the cibarial pump. The methodology developed permits the study detailed of triatomine feeding behaviour using an artificial feeder as well as on live hosts. T. infestans was the species that fed most rapidly on the two hosts tested (28.03+/-1.6 mg/min for pigeon and 21.33+/-1.7 mg/min for mouse), followed by T. brasiliensis (17.09+/-1.4 mg/min and 13.1+/-1.5 mg/min for pigeon and mouse, respectively) and T. pseudomaculata, (5.23+/-0.6 mg/min and 4.09+/-0.4 mg/min for pigeon and mouse, respectively). The quantity of liquid ingested per stroke of the cibarial pump was 100.7+/-4.1 nl for T. infestans, 69.1+/-2.7 nl for T. brasiliensis and 26.8+/-1.5 nl for T. pseudomaculata. The rate of engorgement in pigeons tended to be greater than that obtained for mice in the three species studied. In the experiments carried out using mice, probing times tended to be longer and interruptions during the meal more prolonged. This reinforces the idea that feeding on mice is more difficult than on pigeons, requiring more contact time to obtain the similar quantity of blood.

First report of infection of Lutzomyia longipalpis by Leishmania (Leishmania) infantum from a naturally infected cat of Brazil.

In recent years, cases of feline visceral leishmaniasis (FVL) have been described in different countries. In urban areas, domestic cats are suggested as possible alternative reservoirs of Leishmania (L.) infantum, the causal agent of visceral leishmaniasis (VL). This paper reports the first case of infection of Lutzomyia longipalpis by L. infantum of a naturally infected cat from Brazil through xenodiagnosis. The presence of a cat with FVL and its infectivity to the natural vector in Belo Horizonte city, an endemic area of VL in Brazil, suggests the need for further studies to determine the rate of occurrence of FVL among domestic cats and the infectivity ratio of L. longipalpis in endemic areas, and what is the role of these animals in the epidemiology of the disease.

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs

Summary A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.

Efficacy of Combined Therapy with Liposome-Encapsulated Meglumine Antimoniate and Allopurinol in Treatment of Canine Visceral Leishmaniasis

ABSTRACT An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.