Jorge Luis Chacón

Molecular Characterization of Infectious Bronchitis Virus Strains Isolated from the Enteric Contents of Brazilian Laying Hens and Broilers

Abstract Infectious bronchitis virus (IBV) is the causative agent of avian infectious bronchitis, which is characterized by respiratory, reproductive, and renal signs. However, the role of IBV as an enteric pathogen in still controversial. In Brazil, antigenic groups of IBV divergent from the Massachusetts serotype used for vaccination schedules in that country have already been demonstrated. The present study aimed to assess the different genotypes of IBV in Brazilian commercial poultry flocks by partial sequencing of the S1 amino-terminus coding region using enteric contents as samples and examine their relationship with the vaccine serotype currently in use. Samples of enteric contents were taken as pools of five birds from each of 18 poultry farms (17 broiler and one laying farm) from five Brazilian states between 2002 and 2006. Birds were presenting watery diarrhea and poor general condition but were without respiratory, renal, or reproductive signs. Conventional antibacterial and anticoccidial therapies were unsuccessful and, furthermore, all samples proved negative for rotavirus, reovirus, and astrovirus. Eleven IBV samples were isolated in embryonated eggs and resulted in S1 sequences. Phylogenetic analysis showed that these segregated into an exclusive cluster, close to serotype D274, but distant from Massachusetts. Mean amino acid identity amongst these Brazilian strains was 94.07%; amongst these and serotypes D274, 4/91, and Massachusetts, mean amino acid identity was 77.17%, 69.94%, and 68.93%, respectively. In conclusion, the presence of genotype variant strains of IBV in Brazilian poultry flocks has been demonstrated and might be the reason for the unsuccessful control of IBV in Brazil. Furthermore, these results also strengthen the implications of IBV in enteric diseases of poultry.

Orchitis in Roosters with Reduced Fertility Associated with Avian Infectious Bronchitis Virus and Avian Metapneumovirus Infections

Abstract The pathogenesis of infection involving both infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) causes reproductive damage in hens after viral replication in the epithelium of the oviduct, resulting in loss of cilia and degeneration and necrosis of the epithelial and glandular cells. Although IBV has been indicated as a possible cause of the formation of calcium stones in the epididymus of roosters, a definitive association has not been confirmed. This report describes the detection of IBV and aMPV in the testes of roosters from a Brazilian poultry broiler breeders flock with epididymal stones and low fertility. Samples of testis, trachea, and lungs from breeder males aged 57 wk were positive for IBV by reverse transcriptase–polymerase chain reaction (RT-PCR), and virus isolation and testis samples were also positive for aMPV by RT-PCR. The inoculation of testis samples into embryonated chicken eggs via the allantoic cavity resulted in curled, hemorrhagic, and stunted embryos typical of IBV infection. The allantoic fluid was positive by RT-PCR aimed to amplify the region coding for the S1 subunit of the IBV S gene, but it was not positive for aMPV. Sequence analysis of the amplified fragment revealed a close relationship with European IBV genotype D274, previously unreported in Brazil. These results indicate that IBV and perhaps aMPV are likely to have played a role in the pathogenesis of the testicular disease described and should be regarded as factors that can influence male fertility disease in chickens.

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.

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.

Rotavirus detection and isolation from chickens with or without symptoms

Rotaviruses have been identified as one of the main etiological agents of diarrhea and enteritis in mammals, including humans, and in avian species. Few studies have been published about enteric viruses in Brazilian poultry, including those related to rotavirus infection. Such studies demonstrate significant occurrence and the importance of enteric viruses in poultry presenting intestinal problems. Enteric viruses are the primary cause of injuries to the gut, allowing other agents, especially bacteria, to attach, to penetrate, and to replicate in the enteric tissue, leading to further damage. The aim of the present study was to detect rotavirus in the intestinal contents of layers and broilers by polyacrylamide gel electrophoresis (PAGE) and virus isolation in MA-104 cell culture. A total of 45.3% of all samples were positive to rotavirus; rotavirus frequencies were 48.7% in samples from flocks with diarrhea, 46.4% in flocks with delayed growth, and 30% in asymptomatic flocks. It was possible to isolate rotavirus in MA-104 cells from the nine rotavirus-positive randomly chosen samples. These results indicate that rotavirus may have an important role in pathogenesis of enteric disease.

Emergence of Enteric Viruses in Production Chickens Is a Concern for Avian Health

Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease.

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 São 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.

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 amplification products of 524 bp (external primers) and 219 bp (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 219 bp amplified product was confirmed 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 high 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.

Pathogenicity and molecular characteristics of infectious bronchitis virus (IBV) strains isolated from broilers showing diarrhoea and respiratory disease

Abstract 1. The possibility that infectious bronchitis virus (IBV) variants isolated from broilers with enteric and respiratory problems have a different tropism and pathological outcome from those IBV strains causing classical respiratory disease was investigated. 2. IBV variants were isolated from broiler flocks with enteric and respiratory problems in two regions of Brazil. The USP-10 isolate, of enteric origin, was inoculated via the oral oroculonasal routes into IBV-antibody-free broilers and specific pathogen-free (SPF) chickens to determine tissue tropism and pathogenicity and compared with an IBV variant (USP-50) isolated from chickens showing signs of respiratory disease only. 3. Both USP-10 and USP-50 strains caused similar pathological patterns by either route of inoculation. Both variants were detected in respiratory and non-respiratory tissues, including the kidney, intestine and testis. 4. Broilers were more susceptible to infection than SPF chickens, and seroconversion was detected in all of the chicks.

Detection by reverse transcriptase-polymerase chain reaction and molecular characterization of subtype B avian metapneumovirus isolated in Brazil.

Subtype B avian metapneumovirus (aMPV) was isolated and detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in Brazilian commercial laying chicken flocks with no history of vaccination against aMPV and presenting respiratory signs and decreased egg production. RT-PCR results from samples from three affected flocks revealed that the three isolates were subtype B. Partial sequence analysis of the G glycoprotein gene confirmed that the samples belonged to subtype B and were not of the vaccine type. Comparison of nucleotide and amino acid sequences of the G gene of the three Brazilian aMPV samples with subtype B isolates from other countries revealed 95.1% to 96.1% identity. Nucleotide sequences showed 100% identity among the Brazilian subtype B samples and 95.6% identity with the subtype B vaccine strain used in Brazil. This work describes the circulation of subtype B aMPV in Brazil and discusses its importance in terms of disease epidemiology.