Infectious bronchitis virus replication in the chicken embryo related cell line

The susceptibility of the chicken embryo related (CER) cell line to infectious bronchitis virus (IBV M41) was characterized after five consecutive passages in CER cells. Virus replication was monitored by cytopathic effect observation, electron microscopy, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR). At 96 h post-infection (p.i.), the cytopathic effect was graded 75% by cell fusion, rounding up of cells and monolayer detachment, and the electron microscopy image characterized by coronavirus morphology. Cytoplasmic fluorescence was readily observed by from 24 h p.i. onwards, and at all times the respective viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Extra-cellular virus was measured by virus titration performed on chicken kidney cells and embryonated chicken eggs, and respective titres ranged from 4.0 to 6.0 log(10) EID50/ml on embryonated chicken eggs, and from 2.0 to 6.0 log(10) TCID50/ml on both CER cells and chicken kidney cells studied from 24 to 120 h p.i. These results confirmed that the M41 strain replicated well in the CER cell line.

Detection of turkey coronavirus in commercial turkey poults in Brazil

Poult enteritis complex has been incriminated as a major cause of loss among turkey poults in other countries. We have observed this in Brazil, associated with diarrhoea, loss of weight gain and, commonly, high mortality In this study, we have used the reverse transcriptase polymerase chain reaction (RT-PCR) to detect turkey coronavirus (TCoV) in sick poults 30 to 120 days of age from a particular producer region in Brazil. The RT-PCR was applied to extracts of intestine tissue suspensions, and the respective intestinal contents, bursa of Fabricius, faecal droppings and cloacal swabs. Primers were used to amplify the conserved 3' untranslated region of the genome, and the nucleocapsid protein gene of TCoV Histo pathological and direct immunohistochemical examinations were performed to detect TCoV antigen in infected intestine and bursa slides. All the results from stained tissues revealed lesions as described previously for TCoV infection. The direct immunohistochemical positive signal was present in all intestine slides. However, all bursa of Fabricius tissues analysed were negative. RT-PCR findings were positive for TCoV in all faecal droppings samples, and in 27% of cloacal swabs. Finally, the best field material for TCoV diagnosis was faecal droppings and/or intestine suspensions.

Pathology and Tissue Distribution of Turkey Coronavirus in Experimentally Infected Chicks and Turkey Poults

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction

The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.

Using post-release monitoring data to optimize avian reintroduction programs: a 2-year case study from the Brazilian Atlantic Rainforest

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular epidemiology and evolution of avian infectious bronchitis virus

Mutation and recombination processes are involved in the genetic and phenotypic variations of RNA viruses, leading to the emergence of new variant strains, and give rise to virus population diversity to be modeled by the host, particularly by the immune system, as occurred with infectious bronchitis virus (IBV) in chickens. The consequence is a continuous emergence of new IBV variants with regard to pathotypes, serotypes, and protectotypes. Nucleotide sequencing and subsequent genetic analysis of the S1 and N protein gene sequences provide a fast and accurate method to classify and predict IBV genotype, and a powerful instrument to monitor phylogenetic and epidemiological evolution of IBV variants. Despite the use of vaccination programmes, infectious bronchitis has become a serious problem in Brazil. Thus, a significant number of IBV field variants have been identified circulating in the Brazilian commercial poultries between 2000 to 2006 and more recently in Argentina. These viruses seem to be indigenous, because they demonstrated a low genetic relatedness with the majority of the reference strains from North America, Europe and Asia, but were moderately to highly related one to another. In summary, indigenous field IBV variants were evolving and circulating in the field in Brazil and Argentina, and should be considered as initial candidates for protection against current IBV infectious in chickens. However, in vitro and in vivo studies are needed to determine the pathogenicity and immunogenecity of these new isolates, before defining a new vaccine strain.

Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

STUDIES ON THE USE OF A FORMIC-ACID PROPIONIC-ACID MIXTURE (BIO-ADD(TM)) TO CONTROL EXPERIMENTAL SALMONELLA INFECTION IN BROILER-CHICKENS

Experiments were carried out on the antibacterial effects of a commercial formic acid-propionic acid mixture (Bio-add(TM)) against different Salmonella serptj pes. The preparation exerted a strong antibacterial effect on S. typhimurium strain F98 in artificially contaminated feed. After 28 days storage, the bactericidal effect was still considerable. When chickens were reared on feed that had been treated with Bio-add(TM) and artificially contaminated with different serotypes, S. enteritidis, S. typhimurium and S. agona were not isolated from the caecal contents, but S. infantis was. No organisms of this strain were isolated when a lower feed-contamination rate of bacteria was used.

The effects of ochratoxin/aluminosilicate interaction on the tissues and humoral immune response of broilers

This study aimed to evaluate the effect of dietary ochratoxin, in the presence or absence of aluminosilicate, on the histology of the bursa of Fabricius, liver and kidneys, and on the humoral immune response of broilers vaccinated against Newcastle disease virus. The exposure of birds to 2 p. p. m. ochratoxin, in the presence or absence of aluminosilicate, reduced their humoral immune response and the number of mitotic cells in the bursa. The relative weight of the livers of the birds exposed to this toxin was increased and, microscopically, there was hepatocyte vacuolation and megalocytosis with accompanying hyperplasia of the biliary epithelium. The kidneys showed hypertrophy of the renal proximal tubular epithelium, with thickening of the glomerular basement membrane. Aluminosilicate did not ameliorate the deleterious effects of the ochratoxin.

EXAMINATION BY ELISA OF SERA OBTAINED FROM CHICKEN BREEDER AND LAYER FLOCKS SHOWING EVIDENCE OF FOWL TYPHOID OR PULLORUM DISEASE

An indirect ELISA using soluble whole cell antigen was used to screen serum samples obtained from breeder and layer flocks some of which had shown clinical or bacteriological evidence of infection with Salmonella Gallinarum or S. Pullorum. There was good correlation between Salmonella infection and the presence of serum samples showing high optical density (OD) values. Sera from seven flocks showing high values were retested using group D (0-1, 9, 12) lipopolysaccharide (LPS) and g,m-H flagella as detecting antigens. Sera from six flocks produced high OD values with LPS and low values with flagella confirming infection with a non-flagellate, group D Salmonella while one produced high values with both antigens indicating mixed infection with another group D serotype.

Mast cells in the developing avian eye

Mast cells are present in the eye of Gallus domesticus, appearing in the anterior uvea in embryos at stage 39 HH (13th day). In hatching and adult birds, they are present in the sclera, uvea, pectinate Ligament, and conjunctiva. Mast cells are absent in the cornea, retina, and pecten oculi.Maturing mast cells in the anterior eye segment appear as round cells having eccentric nuclei and a few cytoplasmic metachromatic granules, whose fluorescence increases during development. Mature cells are more numerous in late development, and their cytoplasm is rich in metachromatic and intensely fluorescent granules. Ultrastructurally, maturing mast cells display progranules and a few electron dense and homogeneous granules on one side of the cell. Mast cells of adult birds possess homogeneous cytoplasmic granules, some of which display protuberances that penetrate hollows of adjoining granules. Heterogeneous granules exhibiting latticed and mottled patterns are also present. The existence of mast cells in the anterior eye segment indicates that these cells might perform a physiological role during development and in aqueous humor outflow. They might modulate exchanges between blood and aqueous humor through chemical mediators present in their granules. (C) 1996 Wiley-Liss, Inc.

Development of an open case-based decision-support system for diagnosis in oral pathology

Making diagnoses in oral pathology are often difficult and confusing in dental practice, especially for the lessexperienced dental student. One of the most promising areas in bioinformatics is computer-aided diagnosis, where a computer system is capable of imitating human reasoning ability and provides diagnoses with an accuracy approaching that of expert professionals. This type of system could be an alternative tool for assisting dental students to overcome the difficulties of the oral pathology learning process. This could allow students to define variables and information, important to improving the decision-making performance. However, no current open data management system has been integrated with an artificial intelligence system in a user-friendly environment. Such a system could also be used as an education tool to help students perform diagnoses. The aim of the present study was to develop and test an open case-based decisionsupport system.Methods: An open decision-support system based on Bayes' theorem connected to a relational database was developed using the C++ programming language. The software was tested in the computerisation of a surgical pathology service and in simulating the diagnosis of 43 known cases of oral bone disease. The simulation was performed after the system was initially filled with data from 401 cases of oral bone disease.Results: the system allowed the authors to construct and to manage a pathology database, and to simulate diagnoses using the variables from the database.Conclusion: Combining a relational database and an open decision-support system in the same user-friendly environment proved effective in simulating diagnoses based on information from an updated database.

Recommended Guidelines for Submission, Trimming, Margin Evaluation, and Reporting of Tumor Biopsy Specimens in Veterinary Surgical Pathology

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.