Assessing Pathogenicity for Novel Mutation/Sequence Variants: The Value of Healthy Older Individuals

Improvement in DNA technology is increasingly revealing unexpected/unknown mutations in healthy persons and generating anxiety due to their still unknown health consequences. We report a 44-year-old healthy father of a 10-year-old daughter with bilateral coloboma and hearing loss, but without muscle weakness, in whom a whole-genome CGH revealed a deletion of exons 38-44 in the dystrophin gene. This mutation was inherited from her asymptomatic father, who was further clinically and molecularly evaluated for prognosis and genetic counseling (GC). This deletion was never identified by us in 982 Duchenne/Becker patients. To assess whether the present case represents a rare case of non-penetrance, and aiming to obtain more information for prognosis and GC, we suggested that healthy older relatives submit their DNA for analysis, to which several complied. Mutation analysis revealed that his mother, brother, and 56-year-old maternal uncle also carry the 38-44 deletion, suggesting it an unlikely cause of muscle weakness. Genome sequencing will disclose mutations and variants whose health impact are still unknown, raising important problems in interpreting results, defining prognosis, and discussing GC. We suggest that, in addition to family history, keeping the DNA of older relatives could be very informative, in particular for those interested in having their genome sequenced.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

Pathogenicity of Metarhizium anisopliae towards Rhipicephalus (Boophilus) microplus under laboratory and field conditions

Metarhizium anisopliae is one of the most studied agents of biological control of several arthropod plagues, including the cattle tick Rhipicephalus (Boophilus) microplus. Studies have been conducted to assess the fungal complex infection process towards its hosts. To accomplish that, mutant strains overexpressing or lacking assumed determinant genes for the process were constructed over the years. A fundamental experiment to demonstrate a particular gene or set of genes participation is the bioassay. The comparison of bioassays using wild and engineered strains is an essential tool to affirm a given gene is crucial in the process. Therefore, the in vitro bioassays should mimic the results obtained in tests under field conditions. In this study, tests under laboratory and filed conditions were done and a correlation analysis was performed in order to statistically validate in vitro bioassays. Tick egg laying, larvae hatching and host mortality were recorded in each experiment through 21 days, both under laboratory and field conditions. In all cases, M. anisopliae treatments were statistically different from the control treatments. A linear regression analysis was performed between the cases. Laboratory results showed a statistically significant correlation with the field conditions using the Pearson's Correlation Test (P < 0.01 host mortality - 0.969, tick egg laying - 0.977 and larvae hatching - 0.956). These results legitimize the in vitro bioassays and, therefore, constitute them as a valid tool for studying this fungus behavior, so they can be used to infer M. anisopliae response towards R. (Boophilus) microplus.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n=8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n=5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5×108 tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion=12.5 %), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion= 100 %). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc- Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Characterization of atypical Listeria innocua isolated from swine slaughterhouses and meat markets

Atypical Listeria innocua strains presenting phenotypic characteristics similar to those of Listeria monocyto genes were recently isolated from food and the environment. These isolates also tested positive for virulence genes specific to L. monocytogenes. Here we report the isolation of atypical hemolytic L. innocua strains from the environment of pork processing plants in Brazil. The strains were positive for L. monocytogenes virulence genes hly, inlA and inlB by PCR and presented genotypic similarities with human isolates of L. monocytogenes via the AFLP technique using HindIII single enzyme protocol. Phenotypic and genotypic similarities suggest that these atypical L. innocua may be pathogenic strains. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Epidemiological characterization and risk factors associated with leptospirosis in the brazilian semiarid

Alves C. J., Alcino J. F., Farias A. E. M., Higino S. S. S., Santos F. A., Azevedo S. S., Costa D. F. & Santos C. S. A. B. 2012. [Epidemiological characterization and risk factors associated with leptospirosis in the brazilian semiarid.] Caracterizacao epidemiologica e fatores de risco associados a leptospirose em ovinos deslanados do semiarido brasileiro. Pesquisa Veterinaria Brasileira 32(6): 523-528. Universidade Federal de Campina Grande, Centro de Sa de e Tecnologia Rural, Unidade Academica de Medicina Veterinaria, Av. Universitaria s/n(o), Patos, PB 58700-970, Brazil. E-mail: clebertja@uol.com.br The aim of this investigation was to determine the herd-level and animal-level prevalence of leptospirosis in sheep from the semiarid of Paraiba State, Northeast Brazil, as well as to identify risk factors. Blood samples were collected from 1,275 sheep from 117 flocks in 19 counties in the Sertao mesoregion, Paraiba. For the serological diagnosis of leptospirosis the microscopic agglutination test (MAT) using 24 Leptospira spp. serovars as antigens was carried out. Of the 117 flocks studied 33 (28.20%) presented at least one seropositive sheep, and of the 1,275 animals 69 (5.41%) were seropositive with titers ranging from 100 to 3,200. Reactant serovars were Autumnalis (49.30%), Andamana (27.53%), Sentot (17.39%), Whitcomb (4.34%) and Australis (1.44%). Herd size > 48 sheep (odds ratio = 2.26; 95% CI = 1.33-5.07; p = 0.021) and participation in animal exhibits (odds ratio = 9.05; 95% CI = 0.96-85.71; p = 0.055) were identified as risk factors. The need was suggested for studies on the isolation of the agent, characterization of its pathogenicity and its economic impact on sheep flocks of the region, and sanitary control in sheep agglomerations was recommended.

ISOLATION OF MICROSPORUM GYPSEUM IN SOIL SAMPLES FROM DIFFERENT GEOGRAPHICAL REGIONS OF BRAZIL, EVALUATION OF THE EXTRACELLULAR PROTEOLYTIC ENZYMES ACTIVITIES (KERATINASE AND ELASTASE) AND MOLECULAR SEQUENCING OF SELECTED STRAINS

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

A Genetic and Pathologic Study of a DENV2 Clinical Isolate Capable of Inducing Encephalitis and Hematological Disturbances in Immunocompetent Mice

Dengue virus (DENV) is the causative agent of dengue fever (DF), a mosquito-borne illness endemic to tropical and subtropical regions. There is currently no effective drug or vaccine formulation for the prevention of DF and its more severe forms, i.e., dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There are two generally available experimental models for the study of DENV pathogenicity as well as the evaluation of potential vaccine candidates. The first model consists of non-human primates, which do not develop symptoms but rather a transient viremia. Second, mouse-adapted virus strains or immunocompromised mouse lineages are utilized, which display some of the pathological features of the infection observed in humans but may not be relevant to the results with regard to the wild-type original virus strains or mouse lineages. In this study, we describe a genetic and pathological study of a DENV2 clinical isolate, named JHA1, which is naturally capable of infecting and killing Balb/c mice and reproduces some of the symptoms observed in DENV-infected subjects. Sequence analyses demonstrated that the JHA1 isolate belongs to the American genotype group and carries genetic markers previously associated with neurovirulence in mouse-adapted virus strains. The JHA1 strain was lethal to immunocompetent mice following intracranial (i.c.) inoculation with a LD50 of approximately 50 PFU. Mice infected with the JHA1 strain lost weight and exhibited general tissue damage and hematological disturbances, with similarity to those symptoms observed in infected humans. In addition, it was demonstrated that the JHA1 strain shares immunological determinants with the DENV2 NGC reference strain, as evaluated by cross-reactivity of anti-envelope glycoprotein (domain III) antibodies. The present results indicate that the JHA1 isolate may be a useful tool in the study of DENV pathogenicity and will help in the evaluation of anti-DENV vaccine formulations as well as potential therapeutic approaches.

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses. (C) 2011 Elsevier Ltd. All rights reserved.

Molecular Surveillance of the Newcastle Disease Virus in Domestic and Wild Birds on the North Eastern Coast and Amazon Biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.