Glanders Diagnosis in an Asymptomatic Mare from Brazil: Insights from Serology, Microbiological Culture, Mass Spectrometry, and Genome Sequencing.

This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders.

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol.

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi.

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions

Isolamento e caracterizaçäo de genes II da superfamília gp85/sialidases expressos nas formas amastigotas de Trypanosoma cruzi / Isolation and characterization of group II genes from 85/sialidase superfamily expresses at amastigote stage of Trypanosoma cruzi

O Trypanosoma cruzi é o protozoário flagelado causador da Doença de Chagas ou tripanosomíase americana, que atinge milhoes de pessoas na América Latina. Esse microorganismo apresenta um ciclo de vida complexo e o estágio evolutivo amastigota é a forma de replicaçao do parasita encontrada no hospedeiro mamífero. Os amastigotas se caracterizam pela presença cie um flagelo curto e forma oval, e sao fundamentais para a persistência da infecçao pelo T. cruzi no hospedeiro vertebrado. Pouco se sabe sobre a constituiçao bioquímica das formas amastigotas, tendo sido poucos os genes isolados e caracterizados até o presente. No entanto sabe-se que a superfamília gênica das gp85/sialidases, que codificam para antígenos de superfície com papel importante na adesao e invasao do parasita, possui membros que sao expressos também nas formas amastigotas do T. cruzi. No presente trabalho é feita a caracterizaçao de cDNAs de formas amastigotas intracelulares da cepa Sylvio (clone X10/4) do Trypanosoma cruzi. Esses cDNAs foram denominados TcSx e foram isolados com sonda de um gene que codifica uma glicoproteína de superfície de 85 kilodáltons das formas tripomastigotas do parasita, descrito anteriormente na literatura. Os dados mostram que o gene TcSx23 e seus similares (TcSx2, TcSx7, TcSxl8, TcSx19 e TcSx32) constituem novos genes que podem ser inseridos na família 11 das gp85/sialidases. A análise das seqüências de nucleotídeos e de aminoácidos revela diferenças significativas entre TcSx23 e as proteínas SA85 -1, ASP-1 e ASP-2 que sao expressas nas formas amastigotas. O gene TcSx23 é transcrito e traduzido nas formas tripomastigotas e amastigotas gerando peptídeos de -80 e 50 kDa e anticorpos anti-TcSx23 mostram que a localizaçao celular dos epítopos reconhecidos por esses anticorpos difere nas duas formas do parasita, sendo de superfície em tripomastigotas e intracelular nos amastigotas. Alguns motivos peptídicos presentes em membros da família 11 das gp85/sialidases e que desempenham papel de adesinas, por exemplo Tc85 e gp82, estao ausentes em TcSx23. É possível que o polipeptídeo TcSx23 e...(au)