Simultaneous detection of 7 important Rickettsiales pathogens by TaqMan-probe quantitative real-time PCR / 中华流行病学杂志

Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R2 >0.990 0), the minimum detection limit was 10 copies/μl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.

Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1.@*METHODS@#Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.@*RESULTS@#The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T5015) of 61 °C and the half-life at 50 °C (t1/2, 50 °C) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu2+, Ag+, and anionic surfactants. Its kinetic parameters (Km, kcat, and kcat/Km) were 18.7 mmol/L, 222.3 s-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.@*CONCLUSIONS@#The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae) / Filogenia do endosimbionte Wolbachia em Trichogramma, an alpha-proteobacteria (Rickettsiae)

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".

Biofiltration of trimethylamine in biotrickling filter inoculated with Aminobacter aminovorans

Background: Trimethylamine (TMA) is the main responsible for the odor associated with rotting fish and other annoying odors generated in many industrial activities. Biofiltration has proved to be efficient for treating odorous gaseous emissions. The main objective of this work was to determine the removal capacity of TMA of a biotrickling filter inoculated with Aminobacter aminovorans and to evaluate the effect of H2S on its performance. Results: The maximumspecific growth rate ofA. aminovorans in a liquid culture was 0.15 h -1 , witha TMAto biomass yield of 0.10 (g g -1) and a specific consumption rate of 0.062 g·g-1·h-1 . The initial specific consumption rate of TMA was highly influenced by the presence of H2S in liquid culture at concentrations of 20 and 69 ppm in heading space oftheflasks.ABTF inoculatedwithA. aminovorans showedremoval efficiencieshigher than98%ina range ofloading rate of 0.2 to 8 g·m-3·h-1 at empty bed residence time (EBRT) of 85 and 180 s. No effect on the elimination capacity and efficiency was detected when H2S was added at 20 and 50 ppm to the inlet gaseous emission, though the fraction of A. aminovorans measured by qPCR in the biofilm decreased. Conclusions:Abiotrickling filter inoculated with A. aminovorans can remove efficiently the TMA in a gaseous stream. The elimination capacity of TMA can be negatively affected by H2S, but its effect is not notorious when it is forming part of a biofilm, due to its high specific consumption rate of TMA.

Amblyomma sculptum Berlese 1888, Amblyomma ovale Koch 1844 e Amblyomma aureolatum (Pallas 1772) (Acari: Ixodidae): diversidade genética e detecção de riquétsias em diferentes biomas do Brasil / Amblyomma sculptum Berlese 1888, Amblyomma ovale Koch 1844 and Amblyomma aureolatum (Pallas 1772) (Acari: Ixodidae): genetic diversity and detection of rickettsiae in different biomes in Brazil

A febre maculosa (FM) é a principal doença humana associada a carrapatos no Brasil. Amblyomma sculptum,Amblyomma ovale e Amblyomma aureolatum estão entre os principais vetores dos bioagentes da FM no país. Essas espécies têm ampla distribuição no Brasil, mas os casos de FM associados a esses ixodídeos estão restritos a algumas regiões. Sabendo que populações geneticamente distintas de carrapatos podem ter capacidades vetoriais diferentes, o objetivo desta tese foi analisar a diversidade genética e infecção por riquétsias em populações de A. sculptum, A. ovale e A. aureolatum de diferentes biomas do Brasil. As amostras década espécie foram submetidas à extração de DNA, amplificação e sequenciamento de fragmentos dos genes mitocondriais 12S rDNA,16S rDNA, citocromo oxidase subunidade II e da região D-loop para análise dos carrapatos, e fragmentos dos genes gltA, htrA, ompA, ompB e gene D (sca4) para pesquisa de riquétsia. Análises filogenéticas, filogeográficas e populacionais demonstraram que as populações de A. sculptum têm grande diversidade genética no país, porém sem estruturação geográfica ou por biomas, e com indicativo de expansão em algumas populações no Cerrado

Em A. ovale há estruturação populacional em consequência da associação significativa entre as distâncias genéticas e geográficas, com baixo fluxo gênico entre as populações, sugerindo que pode estar ocorrendo um processo de especiação entre as populações do Cerrado e Caatinga e as populações da Mata Atlântica. A. aureolatum tem baixa diversidade genética e ausência de estruturação para região de Mata Atlântica, com indícios de expansão populacional em algumas das áreas analisadas. Nas populações de A. sculptum do Cerrado foram detectadas Rickettsia amblyommatis, Rickettsia felis (primeiro relato dessas riquétsias nesse ixodídeo para o bioma) e Candidatus Rickettsia andeanae; e na Mata Atlântica foi identificada Candidatus Rickettsia asemboensis (primeiro registro nessecarrapato) e R. Felis nesse ixodídeo. Em espécimes de A. ovale da Caatinga foi observada a presença de Rickettsia bellii e Rickettsia sp. cepa Mata Atlântica; no bioma Mata Atlântica também foi encontrada Rickettsia sp. cepa Mata Atlântica alémde Ca. R. asemboensis (primeiro registro nesse artrópode para o Brasil) e R. felis (primeiro relato nesse ixodídeo). A. aureolatum de área endêmica para FM brasileira foi detectado com infecção por R. bellii e em área não endêmica foi identificado com R. felis (primeiro encontro nesse carrapato). A estruturação de A. ovale não influenciou nas riquétsias detectadas. Não foi observada relação entre os haplótipos dos carrapatos analisados e a detecção de riquétsias. (AU)

Caso probable de tifus murino con falla ventilatoria en una adolescente del área urbana de Cali, Colombia / Probable case of murine typhus with respiratory failure in an adolescent from the urban area of Cali, Colombia

El tifus murino es una enfermedad infecciosa de carácter zoonótico causada por Rickettsia typhi . Se presenta el caso de una paciente femenina de 13 años edad, procedente del área urbana de la ciudad de Cali, quien presentó un cuadro febril asociado a taquipnea y exantema maculopapular generalizado en tronco y extremidades. Durante las primeras 48 h en la UCI pediátrica desarrolló rápido deterioro clínico, fiebre persistente, aumento de reactantes de fase aguda, presencia de infiltrados pulmonares intersticiales bilaterales y derrame pleural, requiriendo ventilación mecánica no invasiva. Se inició tratamiento con doxiciclina frente a la sospecha de una posible infección por agentes rickettsiales. Veinticuatro horas más tarde la paciente presentó mejoría clínica, resolución del exantema y retiro de la ventilación no invasiva. Por medio de la prueba de inmunofluorescencia indirecta se evidenciaron títulos de 1:512 frente al grupo del tifus, constatando el diagnóstico probable de tifus murino.

Murine typhus is a zoonotic infectious disease caused by Rickettsia typhi . We report a case of a 13-year old female patient from the urban area of the city of Cali, who presented with fever, associated with tachypnoea and generalised maculopapular exanthema on the trunk and limbs. During the first 48 h in the paediatric ICU, she rapidly deteriorated, with persistent fever, increased acute phase reactants, bilateral interstitial pulmonary infiltrates and pleural effusion requiring noninvasive ventilation. Treatment with doxycycline was initiated due to a suspected infection by rickettsial agents. Twenty-four hours later the patient presented clinical improvement and resolution of the exanthema, thus the non-invasive ventilation was withdrawn. By means of a indirect immunofluorescence test, titres of 1:512 were shown against the typhus group, leading to the probable diagnosis of murine typhus.

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.

Characterization of intrinsic variability of Mesorhizobium ciceri isolates of cultivated fields.

A large number of putative rhizobial isolates were obtained from the root nodules of various chickpea cultivars growing in agricultural research fields. Of these, thirty were selected and characterized for traits, such as, generation time, intrinsic azide resistance and several symbiotic characters.

Suppression of Meloidogyne javanica by antagonistic and plant growth-promoting rhizobacteria / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Four rhizobacteria selected out of over 500 isolates from rhizosphere of the vegetables in China were further studied for suppression of the root-knot nematode and soil-borne fungal pathogens in laboratory and greenhouse in Belgium. They were identified as Brevibacillus brevis or Bacillus subtilis by Biolog test and partial 16s rDNA sequence comparison. They not only inhibited the radial growth of the root-infecting fungi Rhizoctonia solani SX-6, Pythium aphanidermatum ZJP-1 and Fusarium oxysporum f.sp. cucumerinum ZJF-2 in vitro, but also exhibited strong nematicidal activity by killing the second stage larvae of Meloidogyne javanica to varying degrees in the greenhouse. The toxic principles of bacterium B7 that showed the highest juvenile mortality were partially characterized. The active factors were heat stability and resistance to extreme pH values. B7 used either as seed dressing or soil drench significantly reduced the nematode populations in the rhizosphere and enhanced the growth of mungbean plants over the controls in the presence or absence of R. solani.

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei.

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.

Angiomatosis bacilar / Bacillary angiomatosis

La Angiomatosis Bacilar (AB) es una rara enfermedad infecciosa que afecta piel y vísceras de pacientes con el virus de inmunodeficiencia adquirida y otros inmunodeprimidos. Se caracteriza por presentar lesiones cutáneas vasculares asintomáticas que pueden diseminarse sistémicamente. El agente causal de la angiomatosis bacilar está estrechamente relacionada con la Rocha-limaea henselae