Whole-Genome Sequences of Two Klebsiella pneumoniae Strains (Sequence Types 23 and 35) from Wildlife.

This report describes the draft genomes of two Klebsiella pneumoniae strains that were isolated from two wild boars collected during epidemiological surveillance and monitoring of wild fauna in the Abruzzo and Molise regions. The strains belonged to sequence type 23 (ST23) and ST35, which are frequently reported in clinical cases.

Transcriptomic and computational analysis identified LPA metabolism, KLHL14 and KCNE3 as novel regulators of Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a complex biological program between physiology and pathology. Here, amniotic epithelial cells (AEC) were used as in vitro model of transiently inducible EMT in order to evaluate the transcriptional insights underlying this process. Therefore, RNA-seq was used to identify the differentially expressed genes and enrichment analyses were carried out to assess the intracellular pathways involved. As a result, molecules exclusively expressed in AEC that experienced EMT (GSTA1-1 and GSTM3) or when this process is inhibited (KLHL14 and KCNE3) were identified. Lastly, the network theory was used to obtain a computational model able to recognize putative controller genes involved in the induction and in the prevention of EMT. The results suggested an opposite role of lysophosphatidic acid (LPA) synthesis and degradation enzymes in the regulation of EMT process. In conclusion, these molecules may represent novel EMT regulators and also targets for developing new therapeutic strategies.

Draft Genome Sequences of Mycoplasma mycoides subsp. mycoides Strains APF9 and AP108, Isolated in Nigeria in 2014 to 2016.

Mycoplasma mycoides subsp. mycoides is the etiological agent of contagious bovine pleuropneumonia (CBPP). While several findings on CBPP prevalence in Nigeria were documented, no data were reported about the genomic characterization of Nigerian M. mycoides subsp. mycoides strains. Here, we present the draft genome sequences of two novel M. mycoides subsp. mycoides strains isolated in Nigeria.

MLVA as an Epidemiological Tool To Trace Back Brucella melitensis Biovar 1 Re-Emergence in Italy.

Brucellosis is an important zoonosis caused by Brucella spp., still prevalent in most areas of the world. Brucellosis control in animals is the key to protect humans. The knowledge of Brucella spp. prevailing genotypes in a territory represents an important epidemiological tool to formulate policies and strategies for disease control and to trace back the introduction of new strains previously considered as exotic. In the last years, multiple-locus variable number tandem repeat analysis (MLVA) has been proposed as complementary to classical biotyping methods. MLVA may add important information to the classical epidemiological investigation techniques, to help in tracing back sources of infection in brucellosis outbreaks. Sardinia is an Italian region officially free from sheep and goats brucellosis since 1998. In 2011, Brucella melitensis biovar 1, a biotype not reported in Italy since 1995, was isolated in one flock in the region. The genotyping MLVA-16 showed that isolates belonged to a rare American lineage, confirming it was introduced from other countries. The strain was considered as probably originating from Spain, where this lineage is endemic. BrucellaMLVA-16 has been proved to be useful to analyse the epidemiological correlation of strains enabling to trace its geographic origin by comparing their previously reported genetic patterns.

Whole-Genome Sequencing of Mycoplasma mycoides subsp. mycoides Italian Strain 57/13, the Causative Agent of Contagious Bovine Pleuropneumonia.

Mycoplasma mycoides subsp. mycoides is generally considered one of most pathogenic Mycoplasma species, and it is the etiological agent of contagious bovine pleuropneumonia (CBPP). Here, we present the annotated genome sequence of M. mycoides subsp. mycoides Italian strain 57/13, isolated in 1992 during CBPP outbreaks in Italy.

Choice of next-generation sequencing pipelines.

The next-generation sequencing (NGS) technologies are revolutionary tools which have made possible achieving remarkable advances in genetics since the beginning of the twenty-first century. Thanks to the possibility to produce large amount of sequence data, these tools are going to completely substitute other high-throughput technologies. Moreover, the large applications of NGS protocols are increasing the genetic decoding of biological systems through studies of genome anatomy and gene mapping, coupled to the transcriptome pictures. The application of NGS pipelines such as (1) de-novo genomic sequencing by mate-paired and whole-genome shotgun strategies; (2) specific gene sequencing on large bacterial communities; and (3) RNA-seq methods including whole transcriptome sequencing and Serial Analysis of Gene Expression (Sage-analysis) are fundamental in the genome-wide fields like metagenomics. Recently, the availability of these advanced protocols has allowed to overcome the usual sequencing technical issues related to the mapping specificity over standard shotgun library sequencing, the detection of large structural genomes variations and bridging sequencing gaps, as well as more precise gene annotation. In this chapter we will discuss how to manage a successful NGS pipeline from the planning of sequencing projects through the choice of the platforms up to the data analysis management.

Development and Preliminary Evaluation of a New Real-Time RT-PCR Assay For Detection of Peste des petits Ruminants Virus Genome.

A duplex real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for a simple and rapid diagnosis of Peste des petits ruminants (PPR). qRT-PCR primers and TaqMan probe were designed on a conserved region of nucleocapsid protein (Np) of PPR virus (PPRV) genome. An in vitro transcript of the target region was constructed and tested to determine analytical sensitivity. Commercial heterologous Armored RNA(®) was used as an internal positive control (IPC) for either RNA isolation or RT-PCR steps. The detection limit of the newly designed duplex real-time RT-PCR (qRT-PCR PPR_Np) was approximately 20 copies/µl with a 95% probability. No amplification signals were recorded when the qRT-PCR PPR_Np was applied to viruses closely related or clinically similar to PPRV- or to PPR-negative blood samples. A preliminary evaluation of the diagnostic performance was carried out by testing a group of 43 clinical specimens collected from distinct geographic areas of Africa and Middle East. qRT-PCR PPR_Np showed higher sensitivity than the conventional gel-based RT-PCR assays, which have been used as reference standards. Internal positive control made it possible to identify the occurrence of 5 false-negative results caused by the amplification failure, thus improving the accuracy of PPRV detection.

Molecular typing of Brucella field strains isolated in Italy.

Brucella field strains were identified using molecular techniques. A polymerase chain reaction (PCR) method, based on amplification of the insertion sequence IS711, was used to identify the isolates at species level. Subsequently, restriction fragment length polymorphism analysis of the omp2a and omp2b genes was used to assign the Brucella species to the different biovars. A total of 248 field strains were processed and complete agreement was obtained with the species/biovar identifications made by conventional bacteriological methods. PCR based tests were more rapid and proved valuable in overcoming some of the drawbacks of conventional methods.