Isolation and characterization of polymorphic DNA microsatellite loci for Anopheles triannulatus sensu lato (Diptera: Culicidae) and cross-amplification in congeneric species.

Anopheles (Nyssorhynchus) triannulatus is a complex of 3 species. Thirteen polymorphic microsatellite loci were isolated and characterized in 20 to 25 individuals from Manaus (AM, Brazil). The number of alleles per locus varied from 3 to 10 (mean = 6.0). The observed heterozygosity ranged from 0.250 to 0.875 (mean = 0.680) and expected heterozygosity ranged from 0.376 to 0.844 (mean = 0.698). Two loci exhibited null alleles and all loci were in Hardy-Weinberg equilibrium. No linkage disequilibrium between loci was observed. These loci were used in 4 congeneric species and provide a useful tool for studying population genetics and other aspects of the biology of this and other Anopheles species.

Isolation and characterization of 25 microsatellite DNA loci for Anopheles albitarsis sensu lato and inter-specific amplification in 5 congeneric species.

The Anopheles albitasis complex includes 6 species, and 3 are considered as malaria vectors in Brazil. Twenty-five polymorphic microsatellite DNA loci were isolated and characterized in 24-36 individuals from the neighborhood of Puraquequara, Manaus, Amazonas State, Brazil. The number of estimated alleles ranged from 2 to 10, the observed heterozygosity ranged from 0.182 to 0.897, and the expected heterozygosity ranged from 0.260 to 0.854. Eleven loci showed significant deviation from Hardy-Weinberg equilibrium. Eleven loci were cross-amplified successfully in 5 Anopheles species. These microsatellite loci will be useful in studies investigating population structure and evolutionary genetics in A. albitarsis sensu lato and other A. albitarsis complex species.

Pathological effects of Cryptostegia venusta toxicity in goats and rats.

Here, we aimed to determine the toxicity of Cryptostegia venusta in goats and rats. We orally administered a single 60 g dose of shredded C. venusta leaves per kilogram of body weight to three goats. The animals were necropsied after death, and tissue sections were collected and routinely processed for histopathological analyses. Additionally, we separated 25 adult male Wistar rats (each weighing about 150 g) into five groups: an untreated control group and groups orally treated with 1, 3, 10, or 60 g/kg doses. Rats were sacrificed 72 h after administration of the C. venusta extract, and tissue sections collected for histopathological analyses. All goats presented signs of apathy, salivation, frequent urination, and eventually fatigue 4-6h after receiving C. venusta. Two goats died 20 h after administration, and the third was sacrificed in extremis. The only histopathological finding observed in the goats was lung edema. No rats died during the experimental period or presented any clinical signs or macroscopic lesions. However, both goats and rats exhibited degeneration and multifocal necrosis of cardiac muscle fibers. From our results, we conclude that the C. venusta plant is capable of promoting cardiotoxicity.

Avaliação da atividade cicatrizante do jucá (Caesalpinia ferrea Mart. ex Tul. var. ferrea) em lesões cutâneas de caprinos / Evaluation of the Brazilian ironwood (Caesalpinia ferrea Mart. ex Tul. var. ferrea) healing activity on cutaneous lesions of goats

O objetivo deste estudo foi demonstrar os efeitos do tratamento tópico do jucá (Caesalpinia ferrea) em feridas cutâneas. Quinze caprinos machos sem raça definida foram divididos em 3 grupos de acordo com o pós-cirúrgico (7º, 14º e 21º dias). As feridas experimentais foram tratadas com a pomada composta pela casca da Caesalpinia ferrea em pó misturada com a vaselina estéril e as do grupo controle apenas com a vaselina esterilizada. A aplicação diária da pomada e da vaselina estéril foi realizada sobre ferida circular padronizada de 16 cm² de área na região torácica de cada animal. As avaliações das feridas foram feitas do ponto de vista clínico, bacteriológico, morfométrico e histopatológico nos períodos pré-determinados (7º, 14º e 21º dias). Morfometricamente, as feridas do controle apresentaram áreas cirúrgicas menores e grau de contração maior que as do grupo tratado, entretanto, histologicamente, houve completa epitelização das feridas tratadas no 21º dia, enquanto que as feridas do grupo controle necessitavam de mais tempo para resolução do processo cicatricial. No exame microbiológico realizado no momento da produção da ferida, não se observou crescimento bacteriano e no momento das biópsias, identificou-se a presença de bactérias da família Enterobacteriaceae e Staphylococcus aureus, sendo que a partir do 14º dia observou-se Staphylococcus aureus apenas no grupo controle. A utilização tópica da pomada de Caesalpinia ferrea apresentou eficiência significativa no auxílio da reparação cicatricial de feridas cutâneas de caprinos.

The aim of this study was to demonstrate the effects of the topical treatment with Brazilian ironwood (Caesalpinia ferrea) on cutaneous wounds. Fifteen male mongrel goats were divided into 3 groups according to the postoperative period ((7th, 14th and 21st days). The experimental wounds were treated with an ointment composed of Brazilian wood powder bark mixed with sterile vaseline, whereas controls were only treated with sterile Vaseline. The ointment and the sterile vaselin were daily applied on a standardized circular wound (16 cm² area) in the thoracic region of each animal. Clinical, bacteriological, morphometric and histopathological evaluations were performed in the wounds at predetermined periods (7th, 14th and 21st postoperative days). Morphometrically, control wounds had smaller surgical areas and greater degree of contraction than those from the treated group. However, histologically, there was a complete epithelialization of the treated wounds on the 21st day, whereas control wounds required longer time for healing. In the microbiological evaluation performed at the moment of wound production, there was no bacterial growth. During biopsies, Enterobacteriaceae bacteria and Staphylococcus aureus were identified; from the 14th day, the latter was only observed in the control group. The topical use of Caesalpinia ferrea ointment was significantly efficient to help healing cutaneous wounds in goats.

Avaliação da atividade cicatrizante do óleo de pequi (Caryocar coriaceum Wittm) em feridas cutâneas produzidas experimentalmente em ratos / Evaluation of the healing activity of pequi (Caryocar coriaceum Wittm) oil in cutaneous wounds induced experimentally in rats

O objetivo deste estudo foi demonstrar os efeitos do tratamento tópico do creme à base de óleo de pequi (Caryocar coriaceum Wittm) utilizando 40 ratos (Rattus norvegicus albinus) da linhagem Wistar, machos, com 60 dias de idade. Esses foram divididos em dois grupos: I) composto por 20 ratos com feridas cutâneas tratados com aplicação tópica do creme base com 10% de óleo de pequi; II) com o mesmo número de animais que receberam a aplicação tópica do creme base sem o óleo de pequi. Após antissepsia e anestesia local foi produzida cirurgicamente ferida circular de 1 cm de diâmetro na região dorso lombar. As lesões cutâneas foram avaliadas sob o aspecto clínico, morfométrica e histológico no 3o, 7o, 14o e 21o dias pós-operatório. No grupo tratado com creme à base de óleo de pequi houve aceleração na evolução do processo cicatricial. As feridas dos animais desse grupo apresentaram redução significativa a partir do décimo quarto dia pós-operatório, bem como foram verificados nesse período achados histológicos característicos da etapa final do processo de cicatrização tais como: acentuada quantidade de fibroblastos, fibras colágenas e completo processo de reepitelização, enquanto que as feridas do grupo controle necessitaram de mais tempo para resolução do processo cicatricial.

The main objective of this study was to demonstrate the effects of topical treatment with ointment containing pequi oil (Caryocar coriaceum Wittm), using 40 male 60-day-old mice (Rattus norvegicus albinus) from the Wistar line. These were divided into two groups: I) composed by 20 mice with cutaneous wounds treated by topical application of the ointment based on 10% pequi oil; II) the same number of mice, receiving the topical application of ointment without pequi oil. After antisepsis and local anesthesia, round 1-cm-diameter wounds were made on the lower back region. The wounds were evaluated in regard to clinical, morphometric and histological aspects on the 3rd, 7th, 14th and 21st postoperative days. The group treated with the pequi ointment presented acceleration in the healing process. The animals' wounds of this group showed a meaningful reduction from the 14th postoperative day, when histological characteristics from the ending of the healing process were noted, such as a large amount of fibroblasts, collagen fibers and a complete process of reepithelialization, while the wounds of the control group needed more time for the healing process.

Characterization of spliced leader genes of Trypanosoma ( Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers.

Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage TthII includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

Infection by Trypanosoma vivax in goats and sheep in the Brazilian semiarid region: from acute disease outbreak to chronic cryptic infection.

A study was undertaken to investigate the role of Trypanosoma vivax in sheep and goat mortality and abortions in the Brazilian semiarid region, where outbreaks had been previously reported in bovines. For this purpose, 177 goats and 248 sheep (20% of herds) were randomly sampled on four farms in the State of Paraiba in May and October 2008. The animals were screened for trypanosomes by the buffy coat technique (BCT) and PCR. Infected animals, approximately 25% in both surveys, manifested apathy, pale mucous membranes, enlarged lymph nodes, weakness, weight loss, opacity of the cornea, blindness and abortion. However, the animals with acute and severe disease showing the highest levels of parasitemia and fever, which many times resulted in death, were only detected in the first survey. These severely diseased animals exhibited progressive weight loss and had the smallest packed cell volume (PCV) values. During survey 2, done in October 2008 on the same farms, only animals with low parasitemia and normal temperatures, PCV values and body weights were detected. Therefore, animals that spontaneously recovered from acute infection developed chronic and asymptomatic disease. This finding demonstrated for the first time that sheep and goats, which are the most important livestock in the semiarid region of Brazil, may be severely injured by T. vivax infection and also play a role as asymptomatic carriers and important sources of T. vivax to ruminants in general.

Phylogenetic analysis of Trypanosoma vivax supports the separation of South American/West African from East African isolates and a new T. vivax-like genotype infecting a nyala antelope from Mozambique.

In this study, we addressed the phylogenetic and taxonomic relationships of Trypanosoma vivax and related trypanosomes nested in the subgenus Duttonella through combined morphological and phylogeographical analyses. We previously demonstrated that the clade T. vivax harbours a homogeneous clade comprising West African/South American isolates and the heterogeneous East African isolates. Herein we characterized a trypanosome isolated from a nyala antelope (Tragelaphus angasi) wild-caught in Mozambique (East Africa) and diagnosed as T. vivax-like based on biological, morphological and molecular data. Phylogenetic relationships, phylogeographical patterns and estimates of genetic divergence were based on SSU and ITS rDNA sequences of T. vivax from Brazil and Venezuela (South America), Nigeria (West Africa), and from T. vivax-like trypanosomes from Mozambique, Kenya and Tanzania (East Africa). Despite being well-supported within the T. vivax clade, the nyala trypanosome was highly divergent from all other T. vivax and T. vivax-like trypanosomes, even those from East Africa. Considering its host origin, morphological features, behaviour in experimentally infected goats, phylogenetic placement, and genetic divergence this isolate represents a new genotype of trypanosome closely phylogenetically related to T. vivax. This study corroborated the high complexity and the existence of distinct genotypes yet undescribed within the subgenus Duttonella.

Trypanosomiasis by Trypanosoma vivax in cattle in the Brazilian semiarid: Description of an outbreak and lesions in the nervous system.

An outbreak of trypanosomiasis by Trypanosoma vivax is reported in the semiarid of Paraíba, Northeastern Brazil from May to August 2002. Sixty-four cows out of 130 were affected; 11 died and the other recovered after treatment with diminazene aceturate. Affected animals had fever, anemia, weight loss, hypoglycemia, increased serum levels of aspartate aminotransferase and, in nine cows, nervous signs. All cows with nervous signs died; six of them recovered after treatment, but the disease relapsed. Six cows aborted and one delivered a calf that died immediately after parturition. Thirty-two out of 100 calves were affected and five died. Nervous signs were not observed in the calves. Gross lesions were thickening of the meninges, enlarged lymph nodes and prominent white pulp of the spleen. The main histological lesion was meningoencephalitis and malacia in the brain of cows with nervous signs. No antibodies against trypanosomes were found in 33 blood samples collected before the outbreak in the affected farm and in 29 samples collected at the same time in two other neighbor farms. Until January 2003, all 89 animals tested had antibodies against T. vivax, suggesting the occurrence of sub clinical infections in cattle without clinical signs. Only two out of 85 serum samples collected on April 2004 were positive for T. vivax antibodies. Data obtained suggested that the semiarid region is non-endemic for trypanosomiasis and that disease occurred due to introduction of the parasite in a susceptible population after an apparent rise in the Tabanus spp. population.

Phylogeography of Brachyplatystoma rousseauxii (Siluriformes - Pimelodidae) in the Amazon Basin offers preliminary evidence for the first case of "homing" for an Amazonian migratory catfish.

The large pimelodid, Brachyplatystoma rousseauxii, is one of the two most important catfish species for the fisheries in the Amazon. It is captured by commercial and artisanal fishing fleets in at least five Amazonian countries, at fishing grounds more than 5000 km apart. Current evidence suggests a complex life cycle that includes the longest reproductive migration known for a freshwater fish species. Experimental fisheries have pointed to a decrease in yield in the Western Amazon. However, reliable information about the capture and status of this fishery resource is still nonexistent, and no study has ever addressed its genetic diversity. We sequenced the entire D-loop of 45 individuals of B. rousseauxii, fifteen from each of three different fishing locations along the main channel of the Solimões-Amazonas System covering a distance of around 2200 km. Results of phylogenetic analyses, molecular diversity estimations, analysis of molecular variance, and nested clade analysis, together show that there is no genetic segregation associated with location in the main channel, as one would expect for a migratory species. However, the significant decrease found in genetic diversity towards the western part of the Amazon could be explained by a non-random choice of tributary to spawn. It is possible that the genetic diversity of the migrating schools decreases towards the west because portions of the species' genetic diversity are being "captured" by the different effluents, as the fish migrates to spawn in the headwaters. Like the salmon in North America, B. rousseauxii may be returning to their home tributary to spawn.

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa.

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed.

Phylogeography of Brachyplatystoma rousseauxii (Siluriformes - Pimelodidae) in the Amazon Basin offers preliminary evidence for the first case of [quot ]homing[quot ] for an Amazonian migratory catfish

The large pimelodid, Brachyplatystoma rousseauxii, is one of the two most important catfish species for the fisheries in the Amazon. It is captured by commercial and artisanal fishing fleets in at least five Amazonian countries, at fishing grounds more than 5000 km apart. Current evidence suggests a complex life cycle that includes the longest reproductive migration known for a freshwater fish species. Experimental fisheries have pointed to a decrease in yield in the Western Amazon. However, reliable information about the capture and status of this fishery resource is still nonexistent, and no study has ever addressed its genetic diversity. We sequenced the entire D-loop of 45 individuals of B. rousseauxii, fifteen from each of three different fishing locations along the main channel of the Solimoes-Amazonas System covering a distance of around 2200 km. Results of phylogenetic analyses, molecular diversity estimations, analysis of molecular variance, and nested clade analysis, together show that there is no genetic segregation associated with location in the main channel, as one would expect for a migratory species. However, the significant decrease found in genetic diversity towards the western part of the Amazon could be explained by a non-random choice of tributary to spawn. It is possible that the genetic diversity of the migrating schools decreases towards the west because portions of the species' genetic diversity are being [quot ]captured[quot ] by the different effluents, as the fish migrates to spawn in the headwaters. Like the salmon in North America, B. rousseauxii may be returning to their home tributary to spawn.

Central nervous system effects of the crude extract of Erythrina velutina on rodents.

The aqueous extract (AE) of Erythrina velutina prolonged the sleep duration induced by sodium pentobarbital (control: 6.4 +/- 1.2 min; extract 10 mg/kg, 47.1 +/- 3.9 min; extract 100 mg/kg, 109.4 +/- 7.2 min; F = 243, P < 0.001). In the open field, the extract at the doses of 10 and 50 mg/kg did not changed the number of crossings, rearings nor groomings. On the other hand, at the dose of 200 mg/kg it reduced the number of crossings (q = 6.25, P < 0.05) and groomings (q = 3.91, P < 0.05). When exposed during three consecutive days to the open field, the control animals showed habituation for crossings (F = 17.56, P < 0.001) and rearings (F = 14.01, P < 0.001). The same was not true for animals treated with 10 mg/kg of the extract (crossings: F = 3.59, P > 0.05; rearings: F = 3.62, P > 0.05). At the same dose, the extract blocked the acquisition of foot shock memory (P = 0.9219) when compared to the control values (P = 0.0078). Our data showed that the crude extract of Erythrina velutina at lower doses interferes with mnemonic process for different tasks, while at higher doses, the sedative and neuromuscular blocking actions are the main effects.

Analgesic effect of Dioclea grandiflora constituents in rodents.

An aqueous fraction and dioclein (5,2',5'-trihydroxy-6,7-dimethoxyflavanone) obtained from the ethanolic extract of the Dioclea grandiflora were tested for their analgesic effect in mice and rats employing acetic-induced writhing and tail flick tests, respectively. When administered intraperitoneally the two agents exhibited significant analgesic effects. The activity of both dioclein and aqueous fraction apparently involves an opiate-like mechanism, since their analgesic action was attenuated by naloxone pretreatment.