Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3 h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity. (c) 2005 Elsevier Ltd. All rights reserved.

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6 h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Ingestion of saliva during carbohydrate feeding by Lutzomyia longipalpis (Diptera; Psychodidae)

The aim of this study was to obtain experimental evidence that phlebotomine saliva is actually ingested during the carbohydrate ingestion phase (before and after blood digestion). The ingestion of carbohydrate was simulated as it occurs in the field by offering the insects balls of cotton soaked in sucrose, sucrose crystals or orange juice cells. The results obtained here showed that ingestion occurred under each condition investigated, as indicated by the presence of apyrase, an enzyme used as a marker to detect saliva in the insect gut and/or carbohydrate sources. Saliva ingestion by phlebotomine during the carbohydrate ingestion phase is important to explain how it could promote starch digestion and to trigger Leishmania promastigotes to follow a differentiation pathway as proposed previously by some authors.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Asymptomatic dogs are highly competent to transmit Leishmania (Leishmania) infantum chagasi to the natural vector

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n= 24) and asymptomatic (n= 14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p= 0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p= 0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p= 0.027) and was directly correlated with antibody levels (p= 0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas. © 2013 Elsevier B.V.

Alterações histológicas no intestino de fêmeas de carrapatos Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) expostas a diferentes concentrações de selamectina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Assessment of how pregnancy modifies plasma lead and plasma/whole blood lead ratio in ALAD 1-1 genotype women

Pregnant women are one of the most sensitive populations to the toxic effects associated with lead (Pb) exposure. These effects are primarily associated with plasma Pb (Pb-P), which reflects the most rapidly exchangeable fraction of Pb in the bloodstream, and elevated maternal Pb-P may be more relevant to foetal Pb exposure than whole blood Pb (Pb-B). We investigated how pregnancy affects Pb-B, Pb-P and %Pb-P/Pb-B ratios without the influence of the 6-aminolevulinic acid dehydratase (ALAD) G177C polymorphism, which is a major genetic factor influencing Pb-B, Pb-P and %Pb-P/Pb-B ratios. Genotypes for the ALAD G177C polymorphism were determined by PCR and restriction fragment length digestion in nine pregnant and 20 non-pregnant women, aged 18-33, environmentally exposed to Pb. Here, we included only women with ALAD 1-1 genotype. Pb-P and Pb-B were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. We found no differences in Pb-B (P > 0.05). However, pregnant women had a 2-fold increase in Pb-P and a 3-fold increase in %Pb-P/Pb-B (both P < 0.01) compared to nonpregnant women. These alterations in Pb concentrations associated with pregnancy are similar to those associated with different ALAD gene variants. We can now better appreciate how pregnancy affects foetal exposure to Pb without the influence of this important genetic factor.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

Productive performance, nutrient digestion and metabolism of Holstein (Bos taurus) and Nellore (Bos taurus indicus) cattle and Mediterranean Buffaloes (Bubalis bubalis) fed with corn-silage based diets

The objective of this study was to evaluate the productive performance, nutrients digestion and metabolism of three different genetic groups fed with the same diet based on corn silage. 30 heifers in growth were used of three groups of cattle, the following: Nellore (Bos taurus indicus) (n = 10), Holstein (Bos taurus taurus) (n = 10), and Mediterranean buffaloes (Bubalis bubalis) (n = 10). The animals were fed in groups and received the same experimental diet composed of corn silage and concentrate for growing heifers. In the evaluation of animals the performance, consumption and total apparent digestibility of dry matter and nutrients with the aid of internal markers (chromic oxide) and external (iADF), rumen fermentation, excretion of purine derivatives, nitrogen balance and blood metabolites were measured. No differences were observed in animal performance. There were differences in nutrient intake and apparent digestibility of dry matter and nutrients in different groups of cattle. The concentration of ammonia nitrogen (NH3-N) and short chain fatty acids (SCFA) in the rumen were higher and lower, respectively, for the group of buffaloes in relation to other experimental groups evaluated. When considering the excretion of total purine derivatives, buffaloes showed the lowest value compared to other genetic groups evaluated; about 61.76% of the total genetic group Nellore and 57.62% of the total genetic group Holstein with an average of 33.67 mmol/day. For the buffaloes, the excretion of xanthine and hypoxanthine observed was of 5.11% of total purine derivatives. There was a better nitrogen balance (g/day) for groups of Holstein heifers and Nellore in relation to the group of buffalo heifers. There were differences in the concentrations of urea and urea nitrogen in serum and liver enzymes where the buffaloes had higher values in relation at the bovines. There is a great metabolic diversity among the experimental groups evaluated and it was more exacerbated among buffaloes and bovines, when submitted to the same diet and same management conditions. (C) 2011 Elsevier B.V. All rights reserved.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity.

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr &lt;750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr &gt;1000) were &gt;/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented &lt;/=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.