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.

Avian Infectious Bronchitis Virus in Brazil: A Highly Complex Virus Meets a Highly Susceptible Host Population

Infectious bronchitis (IB) is a highly aggressive disease for poultry in terms of symptoms and economic losses, and the control of this disease is difficult if flocks are not protected against type-specific challenges by the Avian infectious bronchitis virus (IBV). This article summarizes data presented by the author at the Workshop on Infectious Bronchitis 2009 on IB and IBV, including future developments on the field.

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.

Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV. (C) 2009 Elsevier Ltd. All rights reserved.

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.

Development and validation of nested-PCR for the diagnosis of clinical and subclinical infectious laryngotracheitis

A standardised nested-PCR method that amplifies a region of the glycoprotein E gene of avian infectious laryngotracheitis virus (ILTV) has been developed for the diagnosis of infection by Gallid herpesvirus 1. The two sets of primers employed produced the expected ampIification products of 524bp(externa I primers) and 219bp (internal primers) in the presence of ILTV DNA, whereas no Such amplicons were obtained with other avian respiratory pathogens or with DNA extracted from the cells of uninfected chickens. The identity of the 219bp amplified product was con firmed by DNA sequencing. The standardised nested-PCR method detected ILTV DNA from trachea, lung, conjunctiva and trigeminal ganglia samples from flocks of birds with and without clinical signs. and showed hi.-h sensitivity (95.4%) and specificity (93.1%) when compared with the reference test involving virus isolation in specific-pathogen-free chicken embryos. The standardised nested-PCR method described may be used to detect clinical and latent ILTV infections, and will be of significant value for both diagnostic and epidemiological Studies. (c) 2008 Elsevier B.V. All rights reserved.

SEROLOGIC SURVEY FOR SELECTED INFECTIOUS DISEASES IN FREE-RANGING BRAZILIAN TAPIRS (TAPIRUS TERRESTRIS) IN THE CERRADO OF CENTRAL BRAZIL

From September 2000 to January 2002, a serologic survey was conducted in a population of free-ranging Brazilian tapirs (Tapirus terrestris) inhabiting Emas National Park and surrounding areas in Goias state. central Brazil, as part of an ecologic study. Ten tapirs were immobilized with a tiletamine-zolazepam combination. and blood samples were collected. All sera were negative for Leptospira spp.. Brucella abortus, and equine infectious anemia; and one of 10 animals was positive for Toxoplasma goudii. This report represents the first serologic survey for selected infectious diseases in a free-ranging population or Brazilians tapirs in central Brazil.

Chronic colitis associated with HIV infection can be related to intraepithelial infiltration of the colon by CD8+T lymphocytes

Gastrointestinal complications in AIDS patients with diarrhoea are common clinical manifestations, frequently diagnosed by colonoscopy as non-specific colitis. We retrospectively study colon biopsies diagnosed as chronic colitis associated with HIV (CCH). Biopsies were sorted as patients with AIDS (serum CD4 < 200 cell/mm(3)) but without any clear infectious process (n = 12) and patients without HIV infection (n = 24). There are low numbers of CD4+ T lymphocytes in lamina propria of AIDS patients, but CD8+ T populations in this area appear to be similar in all studied groups, regardless of HIV infection or laboratory evidence of a specific agent. We found the clear evidence of CD8+ T cells infiltration in colonic mucosa in HIV patients with microscopic colitis. An imbalance of lymphocyte subpopulations in the colon, both in the lamina propria and epithelium, could result in an intraepithelial CD8 infiltration, involved in the pathogenesis of CCH in AIDS patients.

Rotavirus Gastroenteritis in Children in 4 Regions in Brazil: A Hospital-Based Surveillance Study

Background. Rotavirus is a major cause of gastroenteritis in children. Knowledge of rotavirus genotypes is important for vaccination strategies. Methods. During 2005-2006, rotavirus surveillance studies were conducted in Sao Paulo, Salvador, Goiania, and Porto Alegre, Brazil. Stool samples were collected from children <5 years of age who had diarrhea and were screened by the Rotaclone Enzyme Immunoassay for the presence of rotavirus. Confirmed rotavirus-positive samples were characterized for P and G genotypes by reverse-transcriptase polymerase chain reaction. Results. A total of 510 stool samples were collected. Of these, 221 (43.3%) were positive for rotavirus. Overall, G9 was the predominant G type, followed by G2, and G1; P[4] and P[8] were the predominant P types. The most frequent G/P genotype combination detected was G2P[4], followed by G9P[8], G9P[4], and G1P[8]. G2P[4] was the predominant type in Goiania and Salvador; G9P[8] and G1P[8] were predominant in Sao Paulo and Porto Alegre, respectively. Conclusions. The prevalence, seasonality, and genotype distribution of rotavirus infection varied in different regions in Brazil. With immunization programs, continuous monitoring of rotavirus types is important to detect novel and emerging strains.

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-la Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-la NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model. (C) 2011 Elsevier Ltd. All rights reserved.

Quantitative Detection of Enterotoxigenic Bacteroides fragilis Subtypes Isolated from Children with and without Diarrhea

A rapid real-time PCR ( RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.

Hospital-based Surveillance to Evaluate the Impact of Rotavirus Vaccination in Sao Paulo, Brazil

Background: Brazil implemented routine immunization with the human rotavirus vaccine, Rotarix, in 2006 and vaccination coverage reached 81% in 2008 in Sao Paulo. Our aim was to assess the impact of immunization on the incidence of severe rotavirus acute gastroenteritis (AGE). Methods: We performed a 5-year (2004-2008) prospective surveillance at a sentinel hospital in Sao Paulo, with routine testing for rotavirus in all children less than 5 years of age hospitalized with AGE. Genotypes of positive samples were determined by reverse transcription polymerase chain reaction. Results: During the study, 655 children hospitalized with AGE were enrolled; of whom 169 (25.8%) were positive for rotavirus. In the post-vaccine period, a 59% reduction in the number of hospitalizations of rotavirus AGE and a 42.2% (95% confidence interval [CI], 18.6%-59.0%; P = 0.001) reduction in the proportion of rotavirus-positive results among children younger than 5 years were observed, with the greatest decline among infants (69.2%; 95% CI, 24.7%-87.4%; P = 0.004). Furthermore, the number of all-cause hospitalizations for AGE was reduced by 29% among children aged <5 years. The onset and peak incidences of rotavirus AGE occurred 3 months later in the 2007 and 2008 seasons compared with previous years. Genotype G2 accounted for 15%, 70%, and 100% of all cases identified, respectively, in 2006, 2007, and 2008. Conclusions: After vaccine implementation, a marked decline in rotavirus AGE hospitalizations was demonstrated among children younger than 5 years of age, with the greatest reduction in the age groups targeted for vaccination. The predominance of genotype G2P[4] highlights the need of continued postlicensure surveillance studies.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Domestic Cats Constitute a Natural Reservoir of Human Enteropathogenic Escherichia coli Types

Feces of 70 diarrhoeic and 230 non-diarrhoeic domestic cats from Sao Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non-diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae-theta (three strains), eae-kappa (n = 3), eae-alpha 1 (n = 2), eae-iota (n = 2), one eae-alpha 2, eae-beta 1 and eae-eta each, and two were not typeable. The majority of the EPEC isolates adhered to HEp-2 cells in a localized adherence-like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.