Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males.

Entomological surveys in the state of Maranhão have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranhão, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.

Anti-Leishmania titers and positive skin tests in patients cured of kala-azar

Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6 percent) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2 percent) cases. Ten years post-cure, 39 (52 percent) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.

Anti-Leishmania titers and positive skin tests in patients cured of kala-azar.

Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6%) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2%) cases. Ten years post-cure, 39 (52%) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.

Avaliação da coagulação plasmática e plaquetometria em cães não infectados e infectados experimentalmente com Ehrlichia spp / Plasmatic coagulation and platelet count in dogs uninfected and experimentally infected with Ehrlichia spp

Avaliou-se a ocorrência de distúrbios na coagulação plasmática e na plaquetometria de cães infectados por Ehrlichia spp., durante 15 semanas após o contágio. Doze cães, entre machos e fêmeas, nascidos em estação experimental e com idades entre um e dois anos, foram usados no experimento. Nove cães foram infectados experimentalmente com sangue de cão naturalmente portador de Ehrlichia spp. e três foram mantidos como controle. As alterações na coagulação plasmática não diferiram entre cães infectados e não infectados. A plaquetometria oscilou durante as 15 semanas entre 61x10³/μL e 830x10³/μL, e o menor valor médio foi de 113x10³/μL na sexta semana após a infecção. Concluiu-se que a coagulação plasmática não apresentou alterações significativas nas 15 semanas após infecção e que a contagem plaquetária oscilou entre valores normais, elevados e reduzidos durante esse período.

The effect of Ehrlichia spp. in plasma coagulation and platelet count in dogs during 15 weeks after contamination was evaluated. Twelve male and female dogs one-to-two-year-old were born in the experimental station and were used for the experiment. Nine dogs were infected with blood of dogs naturally bearing Ehrlichia spp., and three were kept as controls. The variation of plasma coagulation did not significantly differ between infected and uninfected dogs. The platelet count oscillated during the period from 61x10³/μL to 830x10³/μL, and the lowest mean value was 113 x 10³/μL at the sixth week after contamination in infected dogs. In conclusion, the plasma coagulation did not significantly change and the platelet count oscillated between normal, increased, and reduced values during the first 15 weeks after Ehrlichia spp. contamination in dogs.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis.

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 +/- 0.17). All nine sera from VL patients had such antibody (0.99 +/- 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 +/- 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 +/- 0.14), and in all VL patients (0.65 +/- 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 +/- 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40 percent) of 45 SC subjects (mean absorbance of 0.49 ± 0.17). All nine sera from VL patients had such antibody (0.99 ± 0.21), while 11 (65 percent) of 17 CVL individuals were seropositive (0.46 ± 0.05). Only three (12 percent) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58 percent) of 45 SC patients (0.35 ± 0.14), and in all VL patients (0.65 ± 0.29). These antibodies were also detected in 13(76 percent) of 17 CVL subjects (0.42 ± 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.

Genetic analysis of multicase families of visceral leishmaniasis in northeastern Brazil: no major role for class II or class III regions of HLA.

Familial aggregation, high relative risk to siblings, and segregation analysis, suggest genetic control of visceral leishmaniasis in Brazil. Class II gene effects in mice, and high circulating tumour necrosis factor alpha in humans, provide reasons to target HLA. Fifteen polymorphic markers across 1.03 Mb (DQB1 to TNFa) were genotyped (87 multicase families; 638 individuals). Model-based parametric analyses using single-point combined segregation and linkage in COMDS, or multi-point linkage in ALLEGRO, failed to detect linkage. Model-free nonparametric affected sibling pair (SPLINK) or NPL(all) score (ALLEGRO) analyses also failed to detect linkage. Information content mapping confirmed sufficient marker information to detect linkage. Analysis of simulated data sets demonstrated that these families had 100% power to detect NPL(all) scores of 5 to 6 (>LOD4; P < 0.00001) over the range (7% to 61%) of age-related penetrances for a disease susceptibility gene. The extended transmission disequilibrium test (TDT) showed no consistent allelic associations between disease and the 15 loci. TDT also failed to detect significant associations between extended haplotypes and disease, consistent with failure to detect significant linkage disequilibrium across the region. Linkage disequilibrium between adjacent groups of markers (HLADQ/DR; 82-1/82-3/-238bpTNFA; LTA/62/TNFa) was not accompanied by significant global haplotype TDT associations with disease. The data suggest that class II/III regions of HLA do not contain major disease gene(s) for visceral leishmaniasis in Brazil.

Genetic epidemiology of visceral leishmaniasis in northeastern Brazil.

Familial clustering of disease, racial differences in asymptomatic:disease ratios, and studies of mice all point to a genetic component for disease susceptibility in visceral leishmaniasis. Analysis of 87 multi-case pedigrees (824 individuals; 138 nuclear families) from a region of northeastern Brazil endemic for Leishmania chagasi demonstrates a high relative risk ratio (lambda(2S) = 34) to further siblings of affected sibling pairs. Complex segregation analysis using POINTER and COMDS show that all single locus models, as well as polygenic and multifactorial models, provide a significantly (P < 0.001) better fit to the data than a sporadic model. Of the genetic models, the general single locus model was not significantly different from additive or dominant single locus models, all of which gave a gene frequency for the putative disease susceptibility allele of approximately 0.002. The general single locus model was strongly favored (P < 0.001) over a recessive single gene model. Using POINTER, polygenic and multifactorial models were clearly rejected (P < 0.001 in all cases) in favor of the general single locus model. Using COMDS, the analysis was extended to consider two locus models. Results under a general two-locus model did not differ significantly from the dominant, additive, or general single locus models. Under this model, one locus was estimated at a gene frequency of 0.0017, i.e., in the same range as the disease susceptibility locus for the most favored single gene models, with the second locus at a much lower frequency of 0.0002. Hence, the data support the hypothesis that a single major gene may be important in determining disease susceptibility in this population. To identify the gene(s) involved, a genome scan with replication using two subsets of these larger pedigrees with power to detect linkage is in progress.

[Visceral leishmaniasis in the State of Maranhão, Brazil: evolution of an epidemic]. / Leishmaniose visceral no estado do Maranhão, Brasil: a evolução de uma epidemia.

The authors provide a brief report on the historical evolution of visceral leishmaniasis in the State of Maranhão, Brazil, evaluating possible factors for growth of the disease in the State and control measures by the Brazilian Ministry of Health to integrate health services into the maintenance of control programs.

Induction and modulation of the immune response to Leishmania by Montenegro's skin test.

The intradermal inoculation in naive or in previously sensitized individuals of small amounts of Leishmania extract (Montenegro's skin test) induced or modulated, respectively, the immune response to Leishmania, as assessed by subsequent Montenegro's skin tests. These phenomena could hinder the interpretation of Montenegro's skin tests in a population already subjected to the test in the past and, in addition, could affect in an unknown way the development of mucosal lesions in people infected with L. braziliensis or L. amazonensis, since those lesions have been associated with hypersensitivity to Leishmania antigens. Anti-Leishmania antibody responses, assessed by enzyme-linked immunosorbent assay, were not induced in naive individuals by Montenegro's skin tests, but tended to become more intense following these tests in previously sensitized individuals.

Holochilus brasiliensis nanus Thomas, 1897. Sugestao de modelo experimental para filariose, leishmaniose e esquistossomose. / Holochilus brasiliensis nanus Thomas, 1897. Suggestion of an experimental model for filariasis, leishmaniasis and schistosomiasis

Roedores silvestres, classificados como Holochilus brasiliensis nanus Thomas, 1897, foram capturados na cidade de Sao Bento, pertencente a Regiao da Baixada, do Estado do Maranhao, Brasil, naturalmente infectados com formas adultas de filaria, na cavidade peritoneal, e microfilarias sanguineas, assim como, com esquistossoma mansoni (vermes adultos e granulomas periovulares hepaticos; intestinais; pulmonares; esplenicos e pancreaticos). Animais nascidos em Bioterio, descendentes de Holochilus da Regiao da Baixada, foram infectados experimentalmente com Leishmania m. amazonensis e Schistosoma mansoni. Em observacoes semanais, foram encontradas lesoes teciduais, semelhantes as que se desenvolvem em hamsters infectados com Leishmania, e hipergamaglobulinemia. Nos esquistossomoticos, foram constatadas hipergamaglobulinemia e reacoes granulomatosas similares as encontradas nos animais infectados naturalmente. Foram observadas, tambem, lesoes hepatica graves, semelhantes as encontradas na esquistossomose humana. Estes achados sugerem a utilizacao do Holochilus b. nanus como modelo experimental destas tres doencas tropicais

Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. An ultrastructural-cytochemical and autoradiographic study.

Partial ligation of the aorta between the renal arteries induces marked atrophy of the cortical tubules of the left (endocrine) kidney with a remarkable increase in the number and granularity of hypersecretory juxtaglomerular cells (JGC), which are found not only at the glomerular pole of arterioles but also in the walls of arteries and arterioles far removed from the glomerulus. Typical vascular smooth muscle cells (SMC), in which secretory granules appear, show a concomitant development of their Golgi complex and rough endoplasmic reticulum, with a gradual decrease in the number of their filaments. Microtubules also appear in the Golgi area. Thiery's periodic acid-thiocarbohydrazide-silver proteinate technique demonstrates that in these "intermediate" cells, as in mature JGC, the amount of glycogen is greater than in SMC. The newly-developed secretory granules of intermediate cells are stained by phosphotungstic acid at a low pH, as are the mature granules of JGC, an indication that both types contain glycoproteins. Light and electron microscopic autoradiography reveal that both JGC and "intermediate" cells of the vascular wall do not incorporate radioactive thymidine (injected during the 10-day observation period). Thus, they develop by metaplasia of preexistent SMC. In control kidneys, radioactive thymidine is practically never incorporated into the nuclei of SMC but is found in a few glomerular and tubular cells of all zones except the papilla.The endocrine kidney shows virtually no reactive nuclei in vascular SMC, glomeruli, or tubular cells of the outer cortex. Thymidine is incorporated into practically all nuclei of the straight portion of proximal tubules and into about half the nuclei of all medullary tubular cells including the papilla.

Ultrastructural cytochemistry of the ischemic (endocrine) kidney.

Partial ligation of the aorta between the renal arteries induces marked atrophy of the cortical tubules (including the macula densa) of the left (endocrine) kidney with a remarkable increase in the number and granularity of hypersecretory juxtaglomerular granulated cells (JGC) which are found not only at the glomerular pole of arterioles but also in the walls of arteries and arterioles far removed from the glomerulus. Staining of fine sections of Araldite-embedded endocrine kidneys according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery reveals abundant glycogen in the JGC and less in the blood vessels and tubules. Juxtaglomerular granules are argentaphobic, but their rim is positively stained when ultrathin sections of glutaraldehyde-fixed, glycol methacrylate-embedded kidneys are exposed to phosphotungstic acid at a low pH. A positive reaction is also shown by the cell coat and lysosomes of JGC as well as by the thickened basal lamina, cell coat, cytosomes, and cytosegresomes of the atrophic tubules. Atrophy is most pronounced in the proximal convoluted tubules, which lose their apical microvilli, their basal infoldings and the majority of their mitochondria and cytosomes.