ABSTRACT: For honey production, beekeepers add one or more supers to the hives to allow honeybees to store their products. However, the increase in hive space can affect the social and health organization in the colony, promoting stress. This study assessed the management of honey production, physicochemical honey properties, population development, and forages immune system gene expression patterns to be used as biomarker for monitoring beekeeping welfare. The treatments comprised 40 beehives divided in four treatments. Treatment 1 - control, supers added according to storage necessity. Treatments 2, 3, and 4 presented two, three, and four supers at the beginning of the experiment, respectively. T1 presented greater honey production (39.4 % increased). No difference in open brood area in the colonies was observed and honey properties and only T2 showed closed brood area higher than the other treatments. Foragers from T4 showed higher catalase and defensin gene expression at the middle-end experiment. Thus, the increasing internal space at the beginning of honey season can affect honey production and immune system of foragers. Catalase and defensin can be used as biomarkers for monitoring honey production welfare.

ABSTRACT Trachoma is a keratoconjunctivitis caused by Chlamydia trachomatis, considered an important leading cause of preventable blindness in the world. This study aimed at verifying if flies can be the vectors for trachoma in our municipality. Flies were assessed in the households of children diagnosed with inflammatory trachoma at the municipality of Botucatu, Sao Paulo State, Brazil. Fly traps were placed in the backyard of the houses during 24 h, in each of the four weather seasons, over a period of one year. The collected dipterans were taxonomically classified and the presence of Chlamydia trachomatis in the flies was evidenced by using the Polymerase Chain Reaction (PCR). During the studied period, 2,188 flies were collected, mainly during the summer and the spring. The most common identified fly was Musca domestica. All fly samples were negative for Chlamydia trachomatis but several other different bacteria were identified in these flies. The authors concluded that flies are probably not the vectors for trachoma in the studied area. Further studies should be conducted to evaluate other possible factors responsible for the maintenance of the disease in our environment.

Abstract Background: The nutritional requirements of honeybees (Apis mellifera) for their complete development need to be supplied through food sources available in the environment, since honeybees are insects that depend directly on blossoming food sources. However, at certain times a food-supply reduction can promote nutritional stress, thus necessitating food supplementation for maintenance or production stimulus of the colonies. Thus, the determination of optimal energy supplementation can assist in the maintenance and production of colonies. Methods: Twenty Apis mellifera beehives were used (with five beehives per treatment): CTL, control (without feeding); SJ, sugarcane juice; SS, sugar syrup; and IS, inverted sucrose. We evaluated the food consumption, population development, and physiological state (expression of vitellogenin and hexamerin 70a genes) of each colony. Results: The results showed that the supplementation of colonies with sugar syrup resulted in an intermediate consumption level (894.6 ± 291 mL) and better development (384.9 ± 237.3 and 158.3 ± 171.6 cm2, sealed and open brood, respectively). Furthermore, this diet ensured that the colonies were in a good physiological state, as bees fed this diet presented the highest relative expression levels of vitellogenin and hexamerin 70a among all the diets tested. Conclusions: Therefore, sugar syrup is concluded to be the best artificial energetic food for use in the supplementation of honeybee colonies.

ABSTRACT Dogs are considered the main reservoir of Leishmania infantum. This protozoan causes visceral leishmaniasis (VL), an uncontrolled urban zoonosis in Brazil. Serological tests and polymerase chain reaction (PCR) on peripheral blood were performed to identify infected dogs in scenarios of higher and lower prevalence of the disease (Teresina and Vitória). One-hundred infected and 57 non-infected animals from Teresina and 100 non-infected animals from Vitória were studied. Animal selection was not dependent on previous serology. The sensitivity (Teresina) and specificity (Teresina and Vitória) were as follows: indirect antibody fluorescence (IFAT) cut-off of 1:40 (IFAT 1:40): 96%, 18%, and 76%; IFAT 1:80: 90%, 33%, and 93%; direct agglutination test (DAT): 96%, 33%, and 98%; fast agglutination screening test (FAST): 93%, 68%, and 100%; immunochromatographic assay with a recombinant rK39 antigen (rK39): 88%, 74%, and 98%; enzyme linked immunosorbent assay (ELISA): 91%, 79%, and 98%; rapid dual-path platform test (TR DPP®): 98%, 60%, and 98%; and blood PCR: 29%, 93%, and 97%, respectively. In the high transmission area, none of the tests adequately discriminated L. infantum-infected from non-infected dogs. However, in the high transmission city, the area under the receiver operating characteristic (ROC) curve of FAST, DAT, ICrK39, ELISA and TR DPP® was high.

IntroductionThe need to control dengue transmission by means of insecticides has led to the development of resistance to most of the products used worldwide against mosquitoes. In the State of São Paulo, the Superintendência de Controle de Endemias(SUCEN) has annually monitored the susceptibility of Aedes aegypti to insecticides since 1996; since 1999, surveys were conducted in collaboration with the National Network of Laboratories (MoReNAa Network) and were coordinated by the Ministry of Health. In this study, in addition to the biological characterization of insecticide resistance in the laboratory, the impact of resistance on field control was evaluated for vector populations that showed resistance in laboratory assays.MethodsField efficacy tests with larvicides and adulticides were performed over a 13-year period, using World Health Organization protocols.ResultsData from the field tests showed a reduction in the residual effect of temephos on populations with a resistance ratio of 3. For adults, field control was less effective in populations characterized as resistant in laboratory qualitative assays, and this was confirmed using qualitative assays and field evaluation.ConclusionsOur results indicated that management of resistance development needs to be adopted when insect populations show reduced susceptibility. The use of insecticides is a self-limiting tool that needs to be applied cautiously, and dengue control requires more sustainable strategies.

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

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.

A pesquisa de infecções por Giardia e a caracterização genotípica deste protozoário foi realizada em primatas não humanos (PNH) mantidos em Zoológico a fim de avaliar o seu potencial zoonótico. As amostras dos animais consistiram de fezes colhidas do piso de 22 baias onde eram mantidos 47 primatas de 18 diferentes espécies. Exames coproparasitológicos foram realizados pelos métodos de concentração por sedimentação e centrífugo-flutuação e revelaram a presença dos seguintes parasitas e suas respectivas frequências: Giardia(18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); oxiurídeos (4.5%) e estrongilídeos (4.5%). O DNA extraído de todas as amostras fecais foi submetido à técnica de PCR para a amplificação dos genes gdh e tpi de Giardia, porém, só foram obtidos amplicons das quatro amostras positivas provenientes de Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. O seqüenciamento dos fragmentos amplificados foi possível apenas para as amostras oriundas de Ateles belzebuth (BA1), Alouatta fusca (BA2) e Alouatta caraya (BA3), cuja análise fenética de ambos os genes revelou pertencerem ao genótipo A. As análises das sequências de tpi revelaram que todas as amostras pertencem ao subgenótipo AII. No que se refere ao gene gdhas análises revelaram uma amostra pertencente ao subgenótipo AII (BA3) e duas ao subgenótipo A1 (BA1 e BA2). Considerando o potencial zoonótico do genótipo A e o fato de que os animais não apresentavam sintomas de infecção, os dados do presente trabalho salientam a importância de se realizar, periodicamente, exames coproparasitológicos dos animais de zoológico, para implementação de medidas preventivas para resguardar a saúde dos animais em cativeiro, a de seus tratadores e dos visitantes de parques zoológicos.

Giardia infections in captive nonhuman primates (NHP) housed at a Brazilian zoo were investigated in order to address their zoonotic potential. Fresh fecal samples were collected from the floors of 22 enclosures where 47 primates of 18 different species were housed. The diagnosis of intestinal parasites after concentration by sedimentation and flotation methods revealed the following parasites and their frequencies: Giardia (18%); Entamoebaspp. (18%); Endolimax nana(4.5%); Iodamoeba spp. (4.5%); Oxyurid (4.5%) and Strongylid (4.5%). Genomic DNA extracted from all samples was processed by PCR methods in order to amplify fragments of gdh and tpi genes of Giardia. Amplicons were obtained from samples of Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. Clear sequences were only obtained for the isolates from Ateles belzebuth (BA1), Alouatta fusca(BA2) and Alouatta caraya (BA3). According to the phenetic analyses of these sequences, all were classified as assemblage A. For the tpi gene, all three isolates were grouped into sub-assemblage AII (BA1, BA2 and BA3) whereas for the gdh gene, only BA3 was sub-assemblage AII, and the BA1 and BA2 were sub-assemblage AI. Considering the zoonotic potential of the assemblage A, and that the animals of the present study show no clinical signs of infection, the data obtained here stresses that regular coproparasitological surveys are necessary to implement preventive measures and safeguard the health of the captive animals, of their caretakers and of people visiting the zoological gardens.

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

We surveyed areas of the state of Rondônia in western Amazon for phlebotomine, which are potential vectors of leishmaniasis. A total of 5,998 specimens were captured, resulting in the identification of 48 species within the Lutzomyia (99.98%) and Brumptomyia (0.02%) genera. The predominant species was Lutzomyia davisi, followed by Lutzomyia umbratilis, Lutzomyia llanosmartinsi, Lutzomyia c. carrerai, Lutzomyia dendrophyla, Lutzomyia nevesi and Lutzomyia whitmani. All sand flies identified as vectors for cutaneous leishmaniasis in Brazil, i.e., Lu. davisi, Lu. umbratilis, Lu. c. carrerai and Lu. whitmani, were found in the surveyed areas.

A presença de infecção por Babesia spp. em cães de áreas rurais do Estado de São Paulo, Brasil foi investigada. Para tanto, 150 cães foram examinados por técnicas parasitológicas de esfregaços sanguíneos e moleculares (PCR), e também, foi verificada a presença de carrapatos nestes animais. Pela análise de esfregaços sanguíneos, 3 (2%) dos cães estavam infectados, enquanto pela PCR, 12 (8%) dos animais foram positivos com bandas aproximadas de 450 pares de base (pb). Foram observados 36 (24%) cães infestados com Rhipicephalus sanguineus ou com Amblyomma spp. As espécies de Amblyomma observadas foram A. cajennense (25%) e A. ovale (25%). Não foi possível correlacionar a presença de R. sanguineus com a infecção por Babesia spp. O seqüenciamento de quatro amostras positivas demonstrou alta identidade com B. canis vogeli, já caracterizada no Brasil.

The status of Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil was studied. For this, 150 animals were examined by blood smears and by PCR; the presence of tick infestation was also investigated. By the blood smear examination, 3 animals (2%) were detected positive and by PCR for Babesia spp. 12 (8%) were positive, with bands visualized in 450 bp. Rhipicephalus sanguineus or Amblyomma spp. were found on 36 (24%) of the 150 dogs. Amblyomma species found were A. cajennense (9/36-25%) and A. ovale (9/36-25%). It was not possible to correlate the presence of R. sanguineus and the infection with Babesia spp. The sequencing of four positive samples demonstrated close identity with B. canis vogeli already characterized in Brazil.

O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia líquida (FPLC) foram analisadas quanto ao perfil eletroforético em géis de poliacrilamida (SDS-PAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina como substrato. A caracterização das enzimas foi realizada a partir da análise do efeito de inibidores sintéticos de cisteína-proteases (E-64, IAA), serina-proteases (PMSF), serina e cisteína-proteases (TPCK, TLCK, elastatinal), metalo-proteases (EDTA) e aspartil proteases (pepstatina) sobre a degradação do substrato. Entre 30 frações eluídas, bandas de proteínas foram observadas em oito delas, entretanto, atividade proteolítica foi detectada apenas nas frações 23, 24, 25 e 26. O perfil eletroforético das proteínas revelou poucas bandas distribuídas na faixa de 45 a 18 kDa. Os zimogramas revelaram zonas de proteólise na faixa de aproximadamente 62 a 35 kDa, entretanto destacaram-se as bandas de hidrólise de 62, 55, 53, 50, 46 e 40 kDa. Nos ensaios de inibição, a proteólise foi marcantemente inibida por E-64, TPCK, TLCK e elastatinal. Redução discreta da proteólise foi observada com IAA e PMSF, enquanto que EDTA e pepstatina não promoveram alteração dos perfis de hidrólise. Estas observações são relevantes, especialmente se considerarmos que para elucidar o envolvimento das proteases na relação parasita-hospedeiro, a purificação dessas moléculas é um requisito importante.

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Longitudinal entomological surveys were performed in Vila Candelária and adjacent rural locality of Bate Estaca concomitantly with a clinical epidemiologic malaria survey. Vila Candelária is a riverside periurban neighborhood of Porto Velho, capital of the state of Rondônia in the Brazilian Amazon. High anopheline densities were found accompanying the peak of rainfall, as reported in rural areas of the region. Moreover, several minor peaks of anophelines were recorded between the end of the dry season and the beginning of the next rainy season. These secondary peaks were related to permanent anopheline breeding sites resulting from human activities. Malaria transmission is, therefore, observed all over the year. In Vila Candelária, the risk of malaria infection both indoors and outdoors was calculated as being 2 and 10/infecting bites per year per inhabitant respectively. Urban malaria in riverside areas was associated with two factors: (1) high prevalence of asymptomatic carriers in a stable human population and (2) high anopheline densities related to human environmental changes. This association is probably found in other Amazonian urban and suburban communities. The implementation of control measures should include environmental sanitation and better characterization of the role of asymptomatic carriers in malaria transmission.