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Parambu, Brazil

Coser J.,Lutheran University of Brazil | Coser J.,University of Cruz Alta | Boeira T.D.R.,Technical Manager | Kazantzi Fonseca A.S.,Simbios Biotecnologia | And 2 more authors.
Brazilian Journal of Infectious Diseases | Year: 2011

Background: It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Objectives: Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Methods: Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. Results: The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. Conclusion: The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing. © 2011 Elsevier Editora Ltda.


Balestrin E.,Lutheran University of Brazil | Fraga A.P.,Lutheran University of Brazil | Ikuta N.,Lutheran University of Brazil | Canal C.W.,Federal University of Rio Grande do Sul | And 2 more authors.
Poultry Science | Year: 2014

Avian infectious bronchitis is a highly contagious viral disease with economic effects on poultry agribusiness. The disease presents multi-systemic clinical signs (respiratory, renal, enteric, and reproductive) and is caused by one coronavirus (infectious bronchitis virus, IBV). Infectious bronchitis virus is classified into different serotypes and genotypes (vaccine strains and field variants). This study aimed to evaluate the occurrence of IBV in commercial poultry flocks from 3 important producing regions in Brazil and to determine the tropism of the main circulating genotypes to 3 different avian physiological systems (respiratory, digestive, urinary/reproductive). Clinical samples with suggestive signs of IBV infection were collected from 432 different poultry commercial flocks (198 from broilers and 234 from breeders). The total number of biological samples consisted of organ pools from the 3 above physiological systems obtained of farms from 3 important producing regions: midwest, northeast, and south. Infectious bronchitis virus was detected by reverse-transcription, real-time PCR of the 5′ untranslated region. The results showed 179 IBV-positive flocks (41.4% of the flocks), with 107 (24.8%) from broilers and 72 (16.8%) from breeders. There were similar frequencies of IBV-positive flocks in farms from different regions of the country, most often in broilers (average 54%) compared with breeders (average 30.8%). reverse-transcription was more frequently detected in the digestive system of breeders (40%), and in the digestive (43.5%) and respiratory (37.7%) systems of broilers. Infectious bronchitis virus genotyping was performed by a reverse-transcription nested PCR and sequencing of the S1 gene from a selection of 79 IBV-positive flocks (45 from broilers and 34 from breeders). The majority of the flocks were infected with Brazilian variant genotype than with Massachusetts vaccine genotype. These results demonstrate the predominance of the Brazilian variant (mainly in the enteric tract) in commercial poultry flocks from 3 important producing regions in Brazil. © 2014 Poultry Science Association Inc.


Fraga A.P.,Lutheran University of Brazil | Fraga A.P.,Grande Rio University | Balestrin E.,Lutheran University of Brazil | Ikuta N.,Lutheran University of Brazil | And 4 more authors.
Avian Diseases | Year: 2013

Infectious bronchitis virus (IBV) is the agent of a highly contagious disease that affects domestic fowl (Gallus gallus). Recent reports showed a high prevalence of one main IBV genotype (Brazil or BR-I) with low genetic diversity in commercial poultry flocks from Brazil. This research analyzed IBV positive poultry flocks from different rearing regions to verify the S1 gene variability and geographic distribution of variant IBV strains in recent years (2010 and 2011). Samples of IBV-positive flocks were obtained from 60 different farms. Forty-nine partial S1 gene sequences were determined and aligned for phylogenetic and amino acid similarity analyses. Eleven samples (22.4%) were similar to Massachusetts vaccine strains (Mass genotype) and 34 samples (69.4%) to the previously characterized Brazilian BR-I genotype. Interestingly, the remaining four samples (8.2%) clustered into a new IBV variant genotype (Brazil-II or BR-II), divergent from the BR-I. A unique nucleotide sequence insertion coding for five amino acid residues was observed in all the Brazilian variant viruses (BR-I and BR-II genotypes). These results show a higher genetic diversity in Brazilian IBV variants than previously described. © American Association of Avian Pathologists.


Fischer C.D.B.,Lutheran University of Brazil | Graf T.,Lutheran University of Brazil | Ikuta N.,Lutheran University of Brazil | Lehmann F.K.M.,Lutheran University of Brazil | And 6 more authors.
Infection, Genetics and Evolution | Year: 2016

Canine distemper virus (CDV) is a highly contagious pathogen for domestic dogs and several wild carnivore species. In Brazil, natural infection of CDV in dogs is very high due to the large non-vaccinated dog population, a scenario that calls for new studies on the molecular epidemiology. This study investigates the phylodynamics and amino-acid signatures of CDV epidemic in South America by analyzing a large dataset compiled from publicly available sequences and also by collecting new samples from Brazil. A population of 175 dogs with canine distemper (CD) signs was sampled, from which 89 were positive for CDV, generating 42 new CDV sequences. Phylogenetic analysis of the new and publicly available sequences revealed that Brazilian sequences mainly clustered in South America 1 (SA1) clade, which has its origin estimated to the late 1980's. The reconstruction of the demographic history in SA1 clade showed an epidemic expanding until the recent years, doubling in size every nine years. SA1 clade epidemic distinguished from the world CDV epidemic by the emergence of the R580Q strain, a very rare and potentially detrimental substitution in the viral genome. The R580Q substitution was estimated to have happened in one single evolutionary step in the epidemic history in SA1 clade, emerging shortly after introduction to the continent. Moreover, a high prevalence (11.9%) of the Y549H mutation was observed among the domestic dogs sampled here. This finding was associated (p < 0.05) with outcome-death and higher frequency in mixed-breed dogs, the later being an indicator of a continuous exchange of CDV strains circulating among wild carnivores and domestic dogs. The results reported here highlight the diversity of the worldwide CDV epidemic and reveal local features that can be valuable for combating the disease. © 2016 Elsevier B.V.


De Carli S.,Lutheran University of Brazil | Ikuta N.,Lutheran University of Brazil | Lehmann F.K.M.,Lutheran University of Brazil | Da Silveira V.P.,Lutheran University of Brazil | And 3 more authors.
Poultry Science | Year: 2015

Escherichia coli is a commensal bacterium of the bird's intestinal tract, but it can invade different tissues resulting in systemic symptoms (colibacillosis). This disease occurs only when the E. coli infecting strain presents virulence factors (encoded by specific genes) that enable the adhesion and proliferation in the host organism. Thus, it is important to differentiate pathogenic (APEC, avian pathogenic E. coli) and nonpathogenic or fecal (AFEC, avian fecal E. coli) isolates. Previous studies analyzed the occurrence of virulence factors in E. coli strains isolated from birds with colibacillosis, demonstrating a high frequency of the bacterial genes cvaC, iroN, iss, iutA, sitA, tsh, fyuA, irp- 2, ompT and hlyF in pathogenic strains. The aim of the present study was to evaluate the occurrence and frequency of these virulence genes in E. coli isolated from poultry flocks in Brazil. A total of 138 isolates of E. coli was obtained from samples of different tissues and/or organs (spleen, liver, kidney, trachea, lungs, skin, ovary, oviduct, intestine, cloaca) and environmental swabs collected from chicken and Turkey flocks suspected to have colibacillosis in farms from the main Brazilian producing regions. Total DNA was extracted and the 10 virulence genes were detected by traditional and/or real-time PCR. At least 11 samples of each gene were sequenced and compared to reference strains. All 10 virulence factors were detected in Brazilian E. coli isolates, with frequencies ranging from 39.9% (irp-2) to 68.8% (hlyF and sitA). Moreover, a high nucleotide similarity (over 99%) was observed between gene sequences of Brazilian isolates and reference strains. Seventy-nine isolates were defined as pathogenic (APEC) and 59 as fecal (AFEC) based on previously described criteria. In conclusion, the main virulence genes of the reference E. coli strains are also present in isolates associated with colibacillosis in Brazil. The analysis of this set of virulence factors can be used to differentiate between APEC and AFEC isolates in Brazil. © 2015 Poultry Science Association Inc.

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