Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Sanitation workers from Portugal: Is there evidence of Leptospira spp?

Leptospires are spirochetes of Leptospira genus. Infection in humans occurs by penetration into the mucous membranes, or into the skin (small wounds or abrasions). Humans are infected when they contact with urine of rodents, the main reservoirs. We aimed to evaluate the presence of anti-Leptospira spp antibodies and leptospiral DNA in sanitation workers (occupational group with increased risk) from Lisbon and Tagus Valley Region (Portugal). Blood samples were collected from 347 sanitation workers, being applied a questionnaire to analyze exposure to rodents and preventive measures. The samples were screened by MACROLepto-test, for the presence of antibodies against pathogenic leptospires. "Positive" and "Non conclusive" samples were then tested with Microscopic Agglutination Test (MAT). Two nested-PCR protocols (primers LeptoA-LeptoB and lipL32) were applied for Leptospira spp DNA detection. It was not observed anti-Leptospira spp antibodies in the worker's samples. However, it was detected non-pathogenic leptospires in a serum sample. Furthermore, 77% had previously seen rodents in the workplace and 94% always used Personal Protective Equipment (PPE). Despite the regular presence of rodents in their workplace, the use of PPE and hygiene measures seemed to be able to prevent the workers contact with this infectious agent.

Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against Moniliophthora perniciosa (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens.

BACKGROUND: Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane-bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. RESULTS: We present the first study aimed at developing novel fungal AOX inhibitors. N-Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j-41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j-41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure-activity relationships analyses. CONCLUSION: We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry.

Sugarcane Cell Wall-Associated Defense Responses to Infection by Sporisorium scitamineum.

The plant cell wall is known to be the first barrier against plant pathogens. Detailed information about sugarcane cell wall-associated defense responses to infection by the causal agent of smut, Sporisorium scitamineum, is scarce. Herein, (immuno)histochemical analysis of two smut resistant and two susceptible sugarcane cultivars was conducted to understand host cell wall structural and compositional modifications in response to fungal infection. Results showed that the fungus grew on the surface and infected the outermost bud scale of both susceptible and resistant cultivars. The present findings also supported the existence of early (24 h after inoculation) and later (72-96 h after inoculation) inducible histopathological responses related to the cell wall modification in resistant cultivars. Lignin and phenolic compounds accumulated during early stages of infection. Later infection response was characterized by the formation of a protective barrier layer with lignin, cellulose and arabinoxylan in the cell walls. Overall, the results suggest possible induction of cell wall-modified responses in smut resistant cultivars to prevent initial entry of the fungus into the meristematic tissues.

Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease.

Currently, direct detection of Leptospira can be done in clinical laboratories by conventional and by real-time PCR (qRT-PCR). We tested a biobank of paired samples of serum and urine from the same patient (202 patients) presenting at the hospital in an area endemic for leptospirosis using qRT-PCR followed by high resolution melting (HRM) analysis. The results were compared with those obtained by conventional nested PCR and with the serologic gold standard microscopic agglutination test (MAT). Differences were resolved by sequencing. qRT-PCR-HRM was positive for 46 of the 202 patients (22.7%, accuracy 100%) which is consistent with known prevalence of leptospirosis in the Azores. MAT results were positive for 3 of the 46 patients (6.5%). Analysis of paired samples allowed us to identify the illness point at which patients presented at the hospital: onset, dissemination or excretion. The melting curve analysis of Leptospira species revealed that 60.9% (28/46) of patients were infected with L. interrogans and 39.1% (18/46) were infected with L. borgpetersenii, both endemic to the Azores. We validated the use of qRT-PCR-HRM for diagnosis of leptospirosis and for identification of the Leptospira species at the earliest onset of infection in a clinical setting, in less than 2 hours.

RNAseq Transcriptional Profiling following Whip Development in Sugarcane Smut Disease.

Sugarcane smut disease is caused by the biotrophic fungus Sporisorium scitamineum. The disease is characterized by the development of a whip-like structure from the primary meristems, where billions of teliospores are produced. Sugarcane smut also causes tillering and low sucrose and high fiber contents, reducing cane productivity. We investigated the biological events contributing to disease symptoms in a smut intermediate-resistant sugarcane genotype by examining the transcriptional profiles (RNAseq) shortly after inoculating the plants and immediately after whip emission. The overall picture of disease progression suggests that premature transcriptional reprogramming of the shoot meristem functions continues until the emergence of the whip. The guidance of this altered pattern is potentially primarily related to auxin mobilization in addition to the involvement of other hormonal imbalances. The consequences associated with whip emission are the modulation of typical meristematic functions toward reproductive organ differentiation, requiring strong changes in carbon partitioning and energy production. These changes include the overexpression of genes coding for invertases and trehalose-6P synthase, as well as other enzymes from key metabolic pathways, such as from lignin biosynthesis. This is the first report describing changes in the transcriptional profiles following whip development, providing a hypothetical model and candidate genes to further study sugarcane smut disease progression.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Complete Genome Sequence of Sporisorium scitamineum and Biotrophic Interaction Transcriptome with Sugarcane.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

First Detection of Borrelia burgdorferi sensu lato DNA in Serum of the Wild Boar (Sus scrofa) in Northern Portugal by Nested-PCR.

Lyme borreliosis is the most common tick-borne zoonosis in the northern hemisphere. Several vertebrates are crucial in the epidemiological cycle of Borrelia burgdorferi sensu lato, but the role of wild boar as a reservoir is still unknown. Sera were collected from 90 wild boars shot in the Trás-os-Montes region, Northern Portugal (hunting season 2011/2012). In this study, Borrelia DNA was detected for the first time by nested-PCR in three different sera, suggesting that the wild boar may be a potential reservoir for this spirochete. Sequencing results show 100% similarity with Borrelia afzelii. Further studies are needed to evaluate the public health risks associated with boar hunting.

SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids.

Many plant species of great economic value (e.g., potato, wheat, cotton, and sugarcane) are polyploids. Despite the essential roles of autopolyploid plants in human activities, our genetic understanding of these species is still poor. Recent progress in instrumentation and biochemical manipulation has led to the accumulation of an incredible amount of genomic data. In this study, we demonstrate for the first time a successful genetic analysis in a highly polyploid genome (sugarcane) by the quantitative analysis of single-nucleotide polymorphism (SNP) allelic dosage and the application of a new data analysis framework. This study provides a better understanding of autopolyploid genomic structure and is a sound basis for genetic studies. The proposed methods can be employed to analyse the genome of any autopolyploid and will permit the future development of high-quality genetic maps to assist in the assembly of reference genome sequences for polyploid species.

First report and differential colonization of Passiflora Species by the B biotype of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in Brazil / Primeiro relato e colonização diferencial de espécies de Passiflora pelo biótipo B de Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) no Brasil

This note is the first report of Bemisia tabaci (Gennadius) biotype B colonizing passionvine in Brazil. We examined the colonization of nine Passiflora species by a wild B type population under greenhouse conditions. P. amethystina Mikan was the most preferred species for oviposition and colonization, whereas P. suberosa L., P. coriacea Juss. and two commercially cultivated species, P. alata Curtis and P. edulis Sims f. flavicarpa Degener, were mostly uncolonised. P. morifolia Mast., P. cincinnata Mast., P. foetida L. and P. caerulea L. showed intermediate levels of colonization. Such differential colonization might suggest some degree of resistance by certain Passiflora species or oviposition preference by B. tabaci.

Esse trabalho descreve pela primeira vez a ocorrência do aleirodídeo Bemisia tabaci (Gennadius) biótipo B colonizando maracujazeiros no Brasil. Também foi examinada a colonização de nove espécies de Passiflora pelo inseto em condições de telado. P. amethystina Mikan foi a espécie de maior preferência para oviposição e colonização, enquanto P. suberosa L., P. coriacea Juss. e duas espécies cultivadas comercialmente, P. alata Curtis e P. edulis Sims f. flavicarpa Degener, foram pouco colonizadas pelo aleirodídeo. P. morifolia Mast., P. cincinnata Mast., P. foetida L. e P. caerulea L. exibiram níveis intermediários de colonização. Esses resultados sugerem que certas espécies de Passiflora exibem diferentes graus de resistência à colonização ou preferência para oviposição de B. tabaci biótipo B.

First report and differential colonization of Passiflora species by the B biotype of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in Brazil.

This note is the first report of Bemisia tabaci (Gennadius) biotype B colonizing passionvine in Brazil. We examined the colonization of nine Passiflora species by a wild B type population under greenhouse conditions. P. amethystina Mikan was the most preferred species for oviposition and colonization, whereas P. suberosa L., P. coriacea Juss. and two commercially cultivated species, P. alata Curtis and P. edulis Sims f. flavicarpa Degener, were mostly uncolonised. P. morifolia Mast., P. cincinnata Mast., P. foetida L. and P. caerulea L. showed intermediate levels of colonization. Such differential colonization might suggest some degree of resistance by certain Passiflora species or oviposition preference by B. tabaci.

Leishmaniose visceral em adolescente gestante / Visceral leishmaniasis in pregnant adolescent

A leishmaniose visceral (LV) é uma doença típica dos países subdesenvolvidos. Atualmente, encontra-se entre as seis endemias consideradas prioritárias no mundo. A cidade de Jacobina, no Estado da Bahia, Brasil, é uma área de elevada endemicidade e, nos últimos anos, vem aumentado o número de casos principalmente na periferia da cidade, atingindo inclusive gestantes. No presente trabalho, é relatado um caso clínico de uma gestante adolescente de 15 anos, portadora de leishmaniose visceral, que não foi tratada durante a gestação, e a criança foi a óbito com dois meses, pesando 1,8 Kg, sem confirmação da transmissão congênita.

Casos de leishmanioses em pacientes atendidos nos Centros de Saúde e hospitais de Jacobina-BA no período de 2000 a 2004 / Cases of leishmaniasis in patients treated in Health Centers and hospitals in Jacobina, Bahia, Brazil, from 2000 to 2004

As leishmanioses são doenças registradas nos continentes asiático, europeu, africano e nas Américas. Originalmente centrada no ambiente silvestre ou em pequenas localidades rurais, passou a ser identificada em centros urbanos, em parte devido à migração do mosquito transmissor (Phlebotominae). No Brasil, constitui-se um grave problema de saúde pública, ocorrendo na maioria dos Estados da federação, principalmente no Nordeste, sendo o município de Jacobina um dos principais focos da doença na Bahia. Com base nos dados da Secretaria Municipal da Saúde, foi realizado um estudo dos casos das leishmanioses nos pacientes atendidos nos Centros de Saúde e hospitais de Jacobina (BA), nos anos de 2000 a 2004. Foi encontrado o registro de 60 casos de leishmaniose tegumentar americana, com maior predisposição para o sexo masculino na faixa etária produtiva, e 45 casos de leishamaniose visceral americana, acometendo principalmente crianças menores de cinco anos. Durante o estudo foi constatada a ocorrência de pacientes atendidos em consultórios particulares, que ainda não tinham sido notificados ao Sistema de Vigilância Epidemiológica.

Leishmaniasis is a disease registered in the continents of Asia, Europe, Africa and America. Originally found in tropical environments or in small rural areas, it has been increasingly 103 found in urban centers, in part due to the migration of the transmitting sand fly (Phlebotominae). In Brazil, this disease constitutes a grave public health problem, occurring in most of the States of the federation, especially in the Northeast. The city of Jacobina represents one of the main focal points of the disease in Bahia. Based on the data from the Municipal Department of Health, a study was conducted with the cases of leishmaniasis in patients treated in health centers and hospitals in the city of Jacobina (BA) from 2000 to 2004. A record of 60 cases of American leishmaniasis tegumentaria were found with a greater predisposition in males in their productive age range, and 45 cases of American visceral leishmaniasis, mainly in children under five years of age. During the study, the occurrence of patients treated in private clinics, which had not yet been registered with the Epidemiologic Surveillance System, was verified.

[Are breastfeeding and complementary feeding of children of adolescent mothers different from those of adult mothers?]. / A amamentação e a alimentação complementar de filhos de mães adolescentes são diferentes das de filhos de mães adultas?

OBJECTIVE: To study breastfeeding during the first year of life and the kind of complementary food provided at one year of life to children of adolescent mothers. To compare these data with breastfeeding and complementary food received by children of adult mothers. METHOD: A dual cohort was performed. Children were selected from the files of CAISM/UNICAMP and assessed when they were one year old. This study consisted of 122 children born from adolescent mothers and 123 children born from adult mothers--full-term births, birthweight was 2,500 g or higher. When the children were one year old, the mothers were interviewed at home or at CIPED/UNICAMP. The results were compared using the chi-square test and the Fisher's test; alpha=5%; the Kaplan-Meier method was used to analyze the duration of breastfeeding and the Wilcoxon test (Breslow) to compare the exclusive, predominant, full and total breastfeeding curves. RESULTS: 94.3% of children of adolescent mothers and 95.9% of children of adult mothers left the maternity hospital being breastfed (p=0.544). The median exclusive breastfeeding duration for both groups was 90 days. After completing one year, 35.3% and 28.5% of children of adolescent and adult mothers, respectively, continued breastfeeding (p=0.254): only breastfeeding 11.5% vs. 8.9% and mixed feeding 23.8% vs. 19.5% (p=0.519). Meat intake by children of adolescent mothers was lower than that of children of adult mothers (13.9% vs. 26.0%; Fisher's test: p=0.031). With regard to egg intake, 11.5% vs. 19.5% of children of adolescent mothers and adult mothers did not eat egg but the results suggested that the egg intake of children of adolescent mothers was higher (p=0.082). CONCLUSION: Duration and pattern of breastfeeding were similar between children of adolescent mothers and of adult mothers. The complementary nutrition was similar, except for a lower intake of meat and a higher intake of eggs among the children of adolescent mothers.

A amamentaçäo e a alimentaçäo complementar de filhos de mäes adolescentes säo diferentes das de filhos de mäes adultas? / Are breastfeeding and complementary feeding of children of adolescentmothers different from those of adult mothers?

OBJETIVOS: estudar a amamentaçäo ao longo do primeiro ano de vida, e o tipo de alimentos complementares utilizados no final do primeiro ano de vida em filhos de mäes adolescentes, e comparar com os filhos de mäes adultas. MÉTODOS: estudo tipo coorte, ambidirecional, no qual as crianças foram selecionadas nos arquivos do CAISM/UNICAMP e avaliadas com um ano de idade. Estudados 122 filhos de adolescentes e 123 filhos de adultas, nascidos a termo, pesando > 2.500 g. Entrevistas realizadas nas casas ou no CIPED/UNICAMP, quando as crianças tinham um ano. Foram usados teste do qui-quadrado e de Fisher, a=5 por cento, análise de sobrevida da amamentaçäo pelo método de Kaplan-Meier e teste de Wilcoxon (Breslow) para comparar as curvas de amamentaçäo exclusiva, predominante, completa e total. RESULTADOS: 94,3 por cento dos filhos de adolescentes e 95,9 por cento dos filhos de adultas saíram da maternidade amamentados (p=0,544). A mediana de amamentaçäo exclusiva foi de 90 dias para ambos os grupos. Com um ano de idade, 35,3 por cento e 28,5 por cento dos filhos de mäes adolescentes e adultas, respectivamente, continuavam amamentados (p=0,254): leite materno 11,5 por cento x 8,9 por cento e aleitamento misto 23,8 por cento x 19,5 por cento (p=0,519). Os filhos de adolescentes ingeriram menos carne diariamente, comparados aos filhos de adultas (13,9 por cento x 26,0 por cento; Fisher: p=0,031). Ovo näo é consumido por 11,5 por cento x 19,5 por cento dos filhos de adolescentes e de adultas, mas sugere ser mais consumido pelos filhos de adolescentes (p=0,082). CONCLUSÕES: O tempo de amamentaçäo e o seu padräo foram semelhantes entre os filhos de mäes adolescentes e de mäes adultas. A alimentaçäo complementar também foi similar, com exceçäo de um menor consumo de carnes e um maior consumo de ovos entre os filhos de mäes adolescentes.