Sergio Ceroni da Silva

Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

The PII Superfamily Revised: A Novel Group and Evolutionary Insights

The PII proteins compose a superfamily of signal transducers with fundamental roles in the nitrogen metabolism of prokaryotic organisms. They act at different cellular targets, such as ammonia transporters, enzymes, and transcriptional factors. These proteins are small, highly conserved, and well distributed among prokaryotes. The current PII classification is based on sequence similarity and genetic linkage. Our work reviewed this classification through an extensive analysis of PII homologues deposited in GenBank. We also investigated evolutionary aspects of this ancient protein superfamily and revised its PROSITE signatures. A new group of PII proteins is described in this work. These PII homologues have a peculiar genetic context, as they are associated with metal transporters and do not contain the canonical PROSITE signatures of PII. Our analysis reveals that horizontal gene transfer could have played an important role in PII evolution. Thus, new insights into PII evolution, a new PII group, and more comprehensive PROSITE signatures are proposed.

Evaluation of PCR Based on Gene apxIVA Associated with 16S rDNA Sequencing for the Identification of Actinobacillus pleuropneumoniae and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.

Detection of Actinobacillus pleuropneumoniae by PCR on field strains from healthy and diseased pigs.

We investigated whether primers able to specifically amplify a 0.7-kb DNA fragment from the conserved cpx genes could be applied to analyze A. pleuropneumoniae field isolates. The specific cpx primers were tested on 120 strains of A. pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to analyze A. pleuropneumoniae isolates from pigs in Brazil. We found that PCR and hybridization were able to discriminate between isolates of A. pleuropneumoniae and other bacteria. The 0.7-kb cpx DNA fragments were amplified from all 63 A. pleuropneumoniae isolates from herds with clinical symptoms and were isolated from lesions of acute cases of swine pleuropneumonia, both serotypable and nonserotypable. The PCR was also applied to 57 field isolates obtained from animals of apparently healthy herds, and the amplified cpx product was present in four serotypable and only two out of eleven A. pleuropneumoniae nonserotypable isolates. All nonserotypable A. pleuropneumoniae isolates revealed the apxA amplification pattern compatible with previously known serotypes. Some nonserotypable isolates might represent a population of isolates that originally were serotypable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes. The PCR method applied is highly sensitive for serotypable A. pleuropneumoniae strains and for nonserotypable strains isolated from acute cases of swine pleuropneumoniae in Brazil.

Genetic variability in Microsporum canis isolated from cats, dogs and humans in Brazil

Dermatophytosis caused by Microsporum canis is a heterogeneous disease with variable clinical manifestations. M. canis is a zoophilic dermatophyte and the most frequent fungi isolated from dogs, cats and children in Brazil. The aim of this study was to investigate the genetic variability of M. canis isolates from different animal species using two microsatellite markers, namely, McGT(13) and McGT(17), and to correlate the results with the clinical and epidemiological patient data in Brazil. The study included a global set of 102 M. canis strains, including 37 symptomatic cats, 35 asymptomatic cats, 19 human patients with tinea, 9 asymptomatic dogs and 2 symptomatic dogs. A total of 14 genotypes were identified, and 6 large populations were distinguished. There was no correlation between these multilocus genotypes and the clinical and epidemiological data, including the source, symptomatology, clinical picture, breed, age, sex, living conditions and geographic location. These results demonstrate that the use of microsatellite polymorphisms is a reliable method for the differentiation of M. canis strains. However, we were unable to demonstrate a shared clinical and epidemiological pattern among the same genotype samples.

Polimorfismos de nucleotídeos únicos em 15 códons do gene da proteína priônica em um rebanho Suffolk afetado com scrapie no Brasil

Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the hosts central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animals genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4% of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96%) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4% in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.

Development of a real-time polymerase chain reaction assay for single nucleotide polymorphism genotyping codons 136, 154, and 171 of the prnp gene and application to Brazilian sheep herds.

Scrapie is a transmissible spongiform encephalopathy of sheep and goats and is associated with the deposition of an abnormal isoform of prion protein (PrPsc). This isoform presents an altered conformation that leads to its aggregation in the host’s central nervous and lymphoreticular systems. A predisposition to the prion-agent infection can be influenced by specific genotypes that are related to polymorphisms in the ovine prnp gene. The most characterized polymorphisms occur at codons 136, 154, and 171, with genotype VRQ being the most susceptible and ARR the most resistant. In the current study, a real-time quantitative polymerase chain reaction (qPCR) technique based on allele-specific TaqMan probes was developed to identify single nucleotide polymorphisms in the prnp gene from Brazilian herds. Specific primers and TaqMan probes were designed for all 3 codons of interest. Samples from a total of 142 animals were analyzed by qPCR, followed by DNA sequencing of the amplicons. All of the genotypes determined by qPCR were in agreement with the data determined by DNA sequencing. In all 3 of the analyzed breeds, the majority of the animals were AA homozygous for the 136 codon. The most frequent genotype for codon 154 was RR, and genotypes QQ and QR were the most frequent for codon 171. The results are discussed in relation to establishing scrapie control measures and breeding programs for Brazilian herds.

Report of outbreaks of classical scrapie in Dorper sheep and associated prion protein gene polymorphisms in affected flocks

Scrapie is an infectious neurodegenerative disease affecting sheep and goats, related with conformational alteration of an isoform of the prion protein that leads to deposition and aggregation in the host’s central nervous system. Occurrence of the natural disease can be influenced by host genetic factors, such as a single nucleotide polymorphism of the prion protein gene. This study reports three scrapie-affected Dorper flocks located on three different farms in Brazil. The objective of this study was to analyze these three flocks using scrapie diagnostics, combining histology, immunohistochemistry, genotyping, and western blot assays. For immunohistochemistry, 192 sheep were selected and 308 sheep blood samples were taken for genotyping. A total of 22 sheep were scrapie positive by immunohistochemistry. Of these, four presented clinical signs and had scrapie immunoreactivity at the obex in western blot assays. The sheep without clinical signs were positive in lymphoid organs, such as the third eyelid and rectal mucosa. The major genotypes found on the flocks were ARQ/ARQ, ARQ/ARR, and ARQ/VRQ for codons 136, 154, and 171. Most of the sheep were considered to be at moderate to high risk, based on risk groups for developing scrapie. Some blood samples were sequenced, and polymorphisms were identified in other codons, such as 127, 142, and 143. Our data demonstrate the importance of preclinical scrapie diagnosis in Brazilian sheep, as most of the affected sheep showed no clinical signs, and emphasize the relevance of genotyping other Dorper sheep to determine the genotypic profile of the breed.

Diagnóstico imuno-histoquímico e caracterização anatomopatológica de Mycoplasma gallisepticum em galinhas de subsistência

Avian mycoplasmosis is caused by bacteria from the Mycoplasmataceae family. Mycoplasma gallisepticum (MG) is the most pathogenic and economically significant species affecting poultry. The aim of this study was to use the immunohistochemistry technique (IHC) as a diagnostic method for the MG infection in poultry. In this report we described two outbreaks of mycoplasmosis caused by MG in free-range chickens. Clinical signs were characterized by prostration, decreased appetite, difficult breathing, nasal and ocular discharge. Necropsy findings were serous secretion in conjunctiva (7/10) and seioses (4/10), edema and caseous exudate; air sacs thickened with foam and caseous exudate (6/10); trachea diffusely reddish (4/10); lungs with 0.5 cm whitish spots (2/10); and pericardial sac with fibrin exudate (2/10). Histologically was observed a lymphoplasmacytic hyperplastic acute tracheitis (10/10), seiositis (5/5) and conjunctivitis (3/4); fibrinonecrotic bronchopneumonia (5/10); acute fibrinous pericarditis (2/10); and fibrinonecrotic aerosaculitis (1/1). IHC anti-MG stained in the extracellular surface of ciliated brush border and/or in the top of epithelium of trachea (10/10), bronchi (5/10) and sinuses (4/5). In seven out of ten cases it was possible to detect MG by real-time PCR from tracheal swabs. IHC anti-MG used as a diagnostic method showed good correlation with clinical signs, lesions and real-time PCR results.

Aspectos fenotípicos, genotípicos e de diagnóstico da bactéria A. pleuropneumoniae

Swine pleuropneumonia is responsible for severe losses in swine breeding farms around the world. The aethiological agent, A. pleuropneumoniae, is classified according to different degrees of pathogenicity in 15 serotypes. The correct identification and serological classification of this bacterium is very important for the adoption of control measures. The diagnosis of swine pleuropneumonia is performed by conventional microbiological techniques, however problems in A. pleuropneumoniae identification have been reported. In Brazil, some studies applying molecular tests have been developed specially in isolates from swine herds with subclinical and chronic infections. In this review, we discuss the results on molecular characterization of A. pleuropneumoniae and related species from herds with or without pleuropneumonia. Moreover, we also discuss the perspectives to development early diagnostic methods and to improve understand of pathogenesis mechanisms of these bacteria.