Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

High-titer antichromatin antibody is associated with proliferative class IV of lupus nephritis

The antichromatin antibody (aCT) has been described as a useful marker for lupus nephropathy. The relevance of its nephritogenic potential may be appropriately evaluated in the context of renal histopathology. Therefore, the present study investigated the relationship of aCT with a particular histopathologic class of lupus nephritis (LN). Seventy-eight consecutive patients with systemic lupus erythematosus (ACR criteria) and active nephritis who underwent renal biopsy from 1999 to 2004 and with available frozen serum sample obtained at the time of biopsy were selected. aCT was measured by ELISA, and anti-dsDNA was measured by indirect immunofluorescence (IIF) and by ELISA. All renal biopsies were revised in a blinded manner by the same expert renal pathologist. Charts were extensively reviewed for demographic and renal features obtained at the time of biopsy. The prevalence of aCT (>= 20 U) was 59% with a mean titer of 74.3 +/- 38.7U. Both aCT-positive and aCT-negative groups of patients had similar age, gender distribution, duration of lupus, and duration of renal disease. Anti-dsDNA was detected by IIF in 29.5% and by ELISA in 42.3% of the patients. Concomitant presence of both antibodies was observed in 63% (29/46) [anti-dsDNA by ELISA] and 45.6% (21/46) [anti-dsDNA by IIF] of the patients. Lower serum levels of C3 (73% vs. 40%, P=0.0058) and C4 (82% vs. 46.7%, P=0.0021) were more commonly observed in aCT >= 20 U patients compared to the aCT-negative group. It is important to note that the use of a higher cut-off value (>= 40 U) for aCT test revealed a predominance of class IV LN (58% vs. 33%, P=0.039) in aCT >= 40 U compared to aCT<40 U group. The mean levels of proteinuria, serum albumin, and creatinine were markedly altered but were comparable in both groups (P >= 0.05). One fourth (26.3%) of the 19 patients with class IV LN and aCT >= 40 U had no detectable anti-dsDNA (ELISA). These data suggest that high-titer aCT seems to be a valuable biomarker for proliferative class IV of LN.

Programa de Controle da Doença de Chagas no Estado de São Paulo: aspectos soroepidemiológicos em microrregiões geográficas homogêneas

A infecção chagásica foi averiguada entre moradores de duas microrregiões geográficas homogêneas do Estado de São Paulo, entre os anos de 1976 a 1980. Campos de Itapetininga, na região de Sorocaba e Encosta Ocidental da Mantiqueira Paulista, na região de Campinas, foram áreas de colonização de Triatoma infestans, no passado, tendo permanecido, na primeira, até o início da década de 70, como reduto da espécie no estado. Atualmente as duas áreas são colonizadas por triatomíneos da espécie Panstrongylus megistus. Perfis de títulos sorológicos caracterizaram ambas as microrregiões como áreas de baixa endemicidade; a interrupção da transmissão foi mais precoce na Encosta, com diferença de 17 anos, em média. Em Campos de Itapetininga, a intensa exposição ao vetor é traduzida pela sororreatividade observada nas idades superiores a 20 anos, correspondentes aos nascidos antes de 1956. Dentre os nascidos entre 1972 e 1977, nessa área, permanece uma baixa positividade, podendo, também, associar-se à transmissão congênita. Na Encosta, a média de idade dos sororreagentes corresponde a nascimentos na década de 1930; os níveis de positividade variaram nos municípios que a compõe segundo o desenvolvimento de capital. Após 1984, com a adoção de novos critérios para o uso da sorologia no Programa de Controle, o encontro de sororreagente não tem sido associado estatisticamente a moradores notificantes de domicílios com presença de triatomíneos.

Serosurvey of antibodies against spotted fever group Rickettsia spp. in horse farms in Northern Paraná, Brazil

Brazilian spotted fever (BSF) is an emerging disease most likely caused by Rickettsia rickettsii. The objective of the present study was to estimate the seroprevalence of BSF rickettsia infections in equines from six horse farms located in Londrina County, Paraná, Southern Brazil. Six owners of horse farms situated in Cambé, Santa Fé, Guaraci and Londrina municipalities participated in the study. All farms were located in areas where BSF has not been reported. A total of 273 horses were sampled and their sera were tested by indirect Immunofluorescence assay (IFA) using R. rickettsii and R. parkeri antigens. Titers equal to and greater than 64 were considered positive. Of 273 sera tested, 15 (5.5%) reacted to R. rickettsii and 5 (1.8%) to R. parkeri. Five out of the six farms studied revealed seropositive animals and seropositivity rate ranged from 0 to 13%. The titers ranged from 64 to 512, and four samples had a titer of 512. Nine animals reacted to R. rickettsii with titers four-fold higher than those for R. parkeri. These results suggest that horses in Northern Paraná may have been exposed to rickettsiae identical or closely related to R. rickettsii.

Prevalence of ehrlichial infection among dogs and ticks in Northeastern Brazil

This study investigated the epidemiology of canine ehrlichiosis in Northeastern Brazil, focusing the identification of the Ehrlichia species and vectors involved. Samples were collected from 472 domestic dogs residing in the health districts of Cajazeiras and Itapuã of Salvador city. The average prevalence of antibodies reactive to E. canis by immunofluorescent antibody test (IFAT) (titer > 1:80) was 35.6% (168/472). Blood samples from the E. canis-seropositive animals were tested by nested PCR in order to identify the Ehrlichia species responsible for the infection. Among the seropositives, 58 (34.5%) were found to be PCR-positive for E. canis. Ticks were found in 32 dogs. Nested-PCR analysis showed that 21.9% (7/32) of the Rhipicephalus sanguineus were infected by E. canis. In both dogs and Rhipicephalus sanguineus, nested-PCR for E. ewingii and E. chaffeensis was negative, with no amplification of DNA fragment.

Anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus) do semiárido paraibano, Nordeste do Brasil

O objetivo do presente trabalho foi determinar a ocorrência de anticorpos anti Brucella abortus, anti Brucella canis e anti Leptospira spp. em raposas (Pseudalopex vetulus). Para tanto, foram utilizadas 60 raposas atropeladas em rodovias no semiárido da Paraíba, Nordeste do Brasil. Para a detecção de anticorpos anti Brucella abortus, o teste do antígeno acidificado tamponado (AAT) foi empregado como teste de triagem, e a prova do 2-mercaptoetanol foi empregada como método confirmatório. Para o diagnóstico sorológico das infecções por Brucella canis e Leptospira spp., foram utilizados os testes de imunodifusão em gel de agar (IDGA) e soroaglutinação microscópica, respectivamente. Todas as amostras foram negativas na pesquisa de anticorpos anti Brucella canis e anti Leptospira spp. Das 60 raposas testadas, 16 (26,6%) foram positivas para anticorpos anti Brucella abortus no teste de AAT, e quatro (6,7%) amostras foram confirmadas no teste de 2-mercaptoetanol, sendo duas amostras com título 100 e duas com título 50.

Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

ISOLATION OF TOXOPLASMA GONDII FROM GOATS FROM BRAZIL

Goats are economically important in many countries, and little is known of caprine toxoplasmosis in Brazil. Anti-bodies to toxoplasma gondii were assayed in the sera of 143 goats from 3 Brazilian states, using modified agglutination test (MAT titer >= 1:25); 46 (32.2%) tested positive. Samples of brain, heart, diaphragm, and masseter of seropositive animals were pooled, digested in pepsin, and bioassayed in mice. Viable T. gondii specimens were isolated from tissue homogenates of 12 goats; the isolates were designated TgGtBr1-12. Ten of the 12 isolates killed 100% of infected mice, indicating that goats can harbor mouse-virulent T. gondii and, hence, can serve as a source of infection for humans.

Tick-Borne Bacteria in Free-Living Jaguars (Panthera onca) in Pantanal, Brazil

Tick-borne bacteria were investigated in 10 free-living jaguars and their ticks in the Pantanal biome, Brazil. Jaguar sera were tested by indirect fluorescent antibody assays using Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia felis, Rickettsia bellii, Ehrlichia canis, and Coxiella burnetii as crude antigens. All 10 jaguar sera reacted (titer >= 64) to at least one Rickettsia species; 4 and 3 sera reacted with E. canis and C. burnetii, respectively. One jaguar presented antibody titer to R. parkeri at least fourfold higher than those to any of the other five Rickettsia antigens, suggesting that this animal was infected by R. parkeri. Ticks collected from jaguars included the species Amblyomma cajennense, Amblyomma triste, and Rhipicephalus (Boophilus) microplus. No Rickettsia DNA was detected in jaguar blood samples, but an A. triste specimen collected on a jaguar was shown by PCR to be infected by R. parkeri. The blood of two jaguars and samples of A. triste, A. cajennense, and Amblyomma sp. yielded Ehrlichia DNA by PCR targeting the ehrlichial genes 16S rRNA and dsb. Partial DNA sequences obtained from PCR products resulted in a new ehrlichial strain, here designated as Ehrlichia sp. strain Jaguar. A partial DNA sequence of the 16S rRNA gene of this novel strain showed to be closest (99.0%) to uncultured strains of Ehrlichia sp. from Japan and Russia and 98.7% identical to different strains of Ehrlichia ruminantium. The ehrlichial dsb partial sequence of strain jaguar showed to be at most 80.7% identical to any Ehrlichia species or genotype available in GenBank. Through phylogenetic analysis, Ehrlichia sp. strain jaguar grouped in a cluster, albeit distantly, with different genotypes of E. ruminantium. Results highlight risks for human and animal health, considering that cattle ranching and ecotourism are major economic activities in the Pantanal region of Brazil.

SEROPREVALENCE AND ISOLATION OF TOXOPLASMA GONDII FROM SHEEP FROM SAO PAULO STATE, BRAZIL

Sheep are important in the epidemiology of Toxoplasma gondii infection, but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 countries of Sao Paulo state. Brazil, using the modified agglutination test (MAT titer >= 1:25); 120 (24.2%) sheep tested positive. Samples of brain, heart, and diaphragm of 85 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenated of 16 sheep, and the isolated were designated TgShBr 1-16. Six of the 16 T. gondii isolated killed 100% of infected mice. Results indicate that asymptomatic sheep can harbour mouse-virulent T. gondii; hence, they can serve as a source of infection for humans.

Occurrence of Neospora caninum antibodies in capybaras (Hydrochaeris hydrochaeris) from Sao Paulo State, Brazil

Capybara (Hydrochaeris hydrochaeris) is a large rodent distributed throughout tropical America. Antibodies to Neospora caninum in 213 feral capybaras from 11 counties of the State of Sao Paulo, Brazil. were assessed using the indirect immunofluorescent antibody test (titer >= 1:25) and found in 20 (9.4%), with titers of 1:25 in 4, 1:50 in 7, and 1:100 in 9 animals. This is the first report of occurrence of N. caninum antibodies in capybaras.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation

Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism. (C) 2010 Elsevier Ltd. All rights reserved.