Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2)

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil. (C) 2007 Elsevier Ltd. All rights reserved.

OCCURRENCE AND MOLECULAR DIAGNOSIS OF CRYPTOSPORIDIUM SERPENTIS IN CAPTIVE SNAKES IN SAO PAULO, BRAZIL

The present study aimed to determine whether Cryptosporidium oocysts were present in stools from captive snakes at Fundacao Parque Zoo logic (Zoological Park Foundation) in Sno Paulo, Brazil. Two collections were performed; the first in July 2008 and the second in February 2009. Fecal samples were collected from 74 enclosures that housed 101 individuals of 23 snake species. The stool specimens collected from 16 out of the 74 enclosures (21.6%) contained Cryptosporidium spp. oocysts; all of them were confirmed as Cryptosporidium serpent is, using molecular techniques. Only in three (18.7%) out of the 16 enclosures with positive samples were there animals with clinical signs compatible with infection by C. serpentis, such as regurgitation and significant progressive weight loss. From the results, it was concluded that diagnostic examinations need to be performed periodically, even on clinically healthy animals, as a preventive measure.

Phenotypic and molecular characterization of recent and archived Erysipelothrix spp. isolated from Brazilian swine

One hundred fifty-one Erysipelothrix spp. isolates from Brazilian swine were characterized by serotyping, determination of antimicrobial susceptibility, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). Among all isolates, 139 were classified in 18 different serotypes and serotype 2b was the most frequent. The susceptibility profiles of the isolates were very similar among each other, which did not permit subtyping Erysipelothrix spp. isolates by the antimicrobial susceptibility testing. Despite the fact that AFLP and PFGE provided the same discriminatory index (0.98), PFGE was more discriminatory than AFLP, given the types of groups it generates. Regardless the technique employed (AFLP or PFGE), no discrimination between recent and historical isolates was established, neither a fixed epidemiologic pattern for their grouping was observed. Nevertheless, AFLP could be an interesting alternative for discriminating the Erysipelothrix species, while PFGE could be an indication for discerning this bacterium according to the serotypes. (C) 2011 Elsevier Inc. All rights reserved.

Rabies virus in insectivorous bats: Implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology

Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil. (C) 2010 Elsevier Inc. All rights reserved.

Molecular Epidemiology of Avian Infectious Bronchitis in Brazil from 2007 to 2008 in Breeders, Broilers, and Layers

Multiple lineages of Brazilian strains from 2007 to 2008 of avian infectious bronchitis virus (IBV) were detected in flocks of breeders, broilers, and layers. Organs samples from 20 IBV-positive flocks with variable clinical signs were submitted to the partial amplification of S gene (nucleotides 726-1071) of IBV. Fifteen of the 20 sequenced strains segregated in a unique Brazilian cluster subdivided in three subclusters (Brazil 01, 02, and 03). Whereas three strains could be classified as Massachusetts (Mass) genotype, the remaining two strains, originating from flocks with reproductive and respiratory disorders, grouped within the 4/91-793B genotype, a genotype that has not been detected before in Brazil. The potential relevance of the findings to the poultry industry is discussed because the low level of identity of the sequenced part of the S gene from 17 of 20 detected field strains and the vaccines of the Massachusetts serotype used suggest that the level of cross-protection by the Massachusetts vaccines might be low.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Physical, epidemiological, and molecular evaluation of infection by Cryptosporidium galli in Passeriformes

Due to the scarcity of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the periodicity of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, and its clinical signs, mortality, and molecular characterization. Four hundred eighty fecal samples were collected from 40 birds, including 372 samples from 31 adult birds and 108 samples from nine young birds (up to 12 months old), housed in five aviaries, monthly from September 2007 to September 2008, with the exception of April. The birds originated from aviaries in which the following species were raised: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Cyanocompsa brissonii), and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate at 4A degrees C until processing. The oocysts were purified by centrifugal flotation in Sheather`s solution, followed by genomic DNA extraction and molecular characterization of oocysts using the nested polymerase chain reaction for amplification of fragments of the 18S subunit of rRNA gene. Intermittent shedding of oocysts was observed by positive amplification for Cryptosporidium spp. in 91 (24.5%) samples of adult birds and 14 (13%) of young birds. The sequencing of the amplified fragments enabled the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in only one aviary and was associated with concomitant infection with Escherichia coli and Isospora sp.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4 degrees C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicurs) and a peach-faced lovebird (Agapornis roseicolis). (C) 2009 Elsevier B.V. All rights reserved.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

Micromorphology of Resin-Dentin Interfaces Using One-Bottle Etch&Rinse and Self-Etching Adhesive Systems on Laser-Treated Dentin Surfaces: A Confocal Laser Scanning Microscope Analysis

Background and Objectives: This study evaluated the hybrid layer (HL) morphology created by three adhesive systems (AS) on dentin surfaces treated with Er:YAG laser using two irradiation parameters. Study Design: Occlusal flat dentin surfaces of 36 human third molars were assigned into nine groups (n = 4) according to the following ASs: one bottle etch&rinse Single Bond Plus (3M ESPE), two-step Clearfil Protect Bond (Kuraray), and all-in-one S3 Bond (Kuraray) self-etching, which were labeled with rhodamine B or fluorescein isothiocyanate dextran and were applied to dentin surfaces that were irradiated with Er:YAG laser at either 120 (38.7 J/cm(2)) or 200 mJ/pulse (64.5 J/cm(2)), or were applied to untreated dentin surfaces (control group). The ASs were light-activated following MI and the bonded surfaces were restored with resin composite Z250 (3M ESPE). After 24 hours of storage in vegetable oil, the restored teeth were vertically, serially sectioned into 1-mm thick slabs, which had the adhesive interfaces analyzed with confocal laser microscope (CLSM-LSM 510 Meta). CLSM images were recorded in the fluorescent mode from three different regions along each bonded interface. Results: Non-uniform HL was created on laser-irradiated dentin surfaces regardless of laser irradiation protocol for all AS, while regular and uniform HL was observed in the control groups. ""Stretch mark""-like red lines were found within the HL as a result of resin infiltration into dentin microfissures, which were predominantly observed in 200 mJ/pulse groups regardless of AS. Poor resin infiltration into peritubular dentin was observed in most regions of adhesive interfaces created by all ASs on laser-irradiated dentin, resulting in thin resin tags with neither funnel-shaped morphology nor lateral resin projections. Conclusion: Laser irradiation of dentin surfaces at 120 or 200 mJ/pulse resulted in morphological changes in HL and resin tags for all ASs evaluated in the study. Lasers Surg. Med. 42:662-670, 2010. (C) 2010 Wiley-Liss, Inc.

Influence of Blood Contamination on Bond Strength of a Self-etching Adhesive to Dental Tissues

Purpose: The aim of this study was to detect the influence of (1) storage period of heparinized blood, (2) type of blood and presence of contaminant, (3) application mode of cleansing agents, and (4) efficacy of cleansing agents on contaminated enamel and dentin during the adhesion process of a one-step adhesive system. Materials and Methods: One hundred four human molars were sectioned into halves along the long axis for enamel and dentin tests. Heparinized and fresh blood were obtained from the same donor, applied and dried to maintain a layer of dry blood on the top of samples. The cleansing agents used were hydrogen peroxide, anionic detergent, and antiseptic solution. A one-step adhesive system (Clearfil S3 Bond) was applied on the dental surface, and composite resin cylinders were built up using Tygon tubing molds. After 24 h, the mu SBS test (1 mm/min) and fracture analysis were performed. Results: There was no statistically significant difference in bond strength values regarding the storage period of heparinized blood and the types of blood. Groups without contamination presented higher bond strengths than contaminated groups. The application mode of the cleansing agents had no influence on bond strength results. There was no statistically significant difference among cleansing agents and they were as effective as a water stream in counteracting the effect of blood contamination. Conclusion: Heparinized blood can be used as a contaminant for up to one week, and it is a reliable procedure to standardize the contaminant. The cleansing agents can be used without friction. A water stream is sufficient to remove blood contamination from dental tissues, before the application of a one-step adhesive system.

Erbium, chromium : yttrium scandium gallium garnet laser for caries removal: influence on bonding of a self-etching adhesive system

This study evaluated the influence of the dental substrates obtained after the use of different caries removal techniques on bonding of a self-etching system. Forty, extracted, carious, human molars were ground to expose flat surfaces containing caries-infected dentine surrounded by sound dentine. The caries lesions of the specimens were removed or not (control-G1) either by round steel burs and water-cooled, low speed, handpiece (G2), or by irradiation with an erbium, chromium:yttrium scandium gallium garnet (Er,Cr:YSGG) laser (2W, 20 Hz, 35.38 J/cm(2), fiber G4 handpiece with 0.2826 mm(2), non-contact mode at a 2 mm distance, 70% air/20% water-G3) or using a chemo-mechanical method (Carisolv-G4). Caries-infected, caries-affected and sound dentines were submitted to a bonding system followed by construction of a resin-based composite crown. Hour-glass shaped samples were obtained and submitted to a micro-tensile bond test. The bond strength data were compared by analysis of variance (ANOVA), complemented by Tukey`s test (P <= 0.05). The samples of sound dentine presented higher bond strengths than did samples of caries-affected dentine, except for the groups treated with the Er,Cr:YSGG laser. The highest bond strengths were observed with the sound dentine treated with burs and Carisolv. The bond strengths to caries-affected dentine were similar in all groups. Additionally, bonding to caries-affected dentine of the Er,Cr:YSGG laser and Carisolv groups was similar to bonding to caries-infected dentine. Thus, caries-affected dentine is not an adequate substrate for adhesion. Moreover, amongst the caries removal methods tested, the Er,Cr:YSGG laser irradiation was the poorest in providing a substrate for bonding with the tested self-etching system.

Deproteinized dentin: A favorable substrate to self-bonding resin cements?

The adhesive performance on deproteinized dentin of different self-adhesive resin cements was evaluated through microtensile bond strength (mu TBS) analysis and scanning electron microscopy (SEM). Occlusal dentin of human molars were distributed into different groups, according to the categories: adhesive cementation with two-step bonding systems-control Groups (Adper Single Bond 2 + RelyX ARC/3M ESPE; One Step Plus + Duolink/Bisco; Excite + Variolink I/Ivoclar Vivadent) and self-adhesive cementation-experimental groups (Rely X Unicem/3M ESPE; Biscem/Bisco; MultiLink Sprint/Ivoclar Vivadent). Each group was subdivided according to the dentin approach to: alpha, maintenance of collagen fibers and beta, deproteinization. The mean values were obtained, and submitted to ANOVA and Tukey test. Statistical differences were obtained to the RelyX Unicem groups (alpha = 13.59 MPa; beta = 30.19 MPa). All the BIS Group specimens failed before the mechanical tests. Dentinal deproteinization provided an improved bond performance for the self-adhesive cement Rely X Unicem, and had no negative effect on the other cementing systems studied. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 98B: 387-394, 2011.