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.

Avian Metapneumovirus Subtypes Circulating in Brazilian Vaccinated and Nonvaccinated Chicken and Turkey Farms

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.

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.

Serosurvey Of Selected Avian Pathogens In Brazilian Commercial Rheas (Rhea americana) And Ostriches (Struthio camelus)

Ratite farming of has expanded worldwide. Due to the intensive farming methods used by ratite producers, preventive medicine practices should be established. In this context, the surveillance and control of some avian pathogens are essential for the success of the ratite industry; however, little is known on the health status of ratites in Brazil. Therefore, the prevalence of antibodies against Newcastle Disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, Mycoplasma synoviae, and Salmonella Pullorum were evaluated in 100 serum samples collected from commercial ostriches and in 80 serum samples from commercial rheas reared in Brazil. All sampled animals were clinically healthy. The results showed that all ostriches and rheas were serologically negative to Newcastle disease virus, Chlamydophila psittaci, Myco plasma gallisepticum, and Myco plasma synoviae. Positive antibody responses against Salmonella Pullorum antigen were not detected in ostrich sera, but were detected in two rhea serum samples. These results can be considered as a warning as to the presence of Salmonella spp. in ratite farms. Therefore, the implementation of good health management and surveillance programs in ratite farms may contribute to improve not only animal production, but also public health conditions.

The visual ecology of avian photoreceptors

The spectral sensitivities of avian retinal photoreceptors are examined with respect to microspectrophotometric measurements of single cells, spectrophotometric measurements of extracted or in vitro regenerated visual pigments, and molecular genetic analyses of visual pigment opsin protein sequences. Bird species from diverse orders are compared in relation to their evolution, their habitats and the multiplicity of visual tasks they must perform. Birds have five different types of visual pigment and seven different types of photo receptor-rods, double (uneven twin) cones and four types of single cone. The spectral locations of the wavelengths of maximum absorbance (lambda (max)) of the different visual pigments, and the spectral transmittance characteristics of the intraocular spectral filters (cone oil droplets) that also determine photoreceptor spectral sensitivity, vary according to both habitat and phylogenetic relatedness. The primary influence on avian retinal design appears to be the range of wavelengths available for vision, regardless of whether that range is determined by the spectral distribution of the natural illumination or the spectral transmittance of the ocular media (cornea, aqueous humour, lens, vitreous humour). Nevertheless, other variations in spectral sensitivity exist that reflect the variability and complexity of avian visual ecology. (C) 2001 Elsevier Science Ltd. All rights reserved.

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand

A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to greater than or equal to 1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00-0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France.

Dietary supplementation with vitamin E modulates avian intestinal immunity

The effect of dietary vitamin E on immunoglobulin A (IgA) antibody production, which acts as the first line of defence at the intestinal mucosa, has not been evaluated in chickens. In the present study the impact of the inclusion of supplementary levels of vitamin E to the diet, on total and antigen-specific IgA antibody titres, T-cell subsets and Ia+ cells, was assessed. From hatching, chickens received a maize-based diet which was supplemented with either 25, 250, 2500 or 5000 mg dl-alpha-tocopherol acetate/kg. Primary immunisation with tetanus toxoid (T. toxoid) emulsified in a vegetable oil-in-water adjuvant was administered by the intraperitoneal route at 21 d of age. At 35 d of age all birds received an oral booster vaccination of T. toxoid. Significantly higher total IgA antibody titres were present in the day 42 intestinal scrapings of birds receiving the 5000 mg/kg vitamin E-supplemented diet (VESD) (P=0.05) and a notable increase was observed in birds receiving the 250 mg/kg VESD (P=0.06). At days 21 and 42 total serum IgA antibody titres of birds receiving the 250 mg/kg VESD was significantly higher (P

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Avian infectious bronchitis virus (IBV): differential pathological and immunological profiles of two Brazilian variants.

2015

Evolution of Teeth and Quadrate in Non-avian Theropoda (Dinosauria: Saurischia), with the Description of Torvosaurus Remains from Portugal

Theropods form a highly successful and morphologically diversified group of dinosaurs that gave rise to birds. They include most, if not all, carnivorous dinosaurs, yet many theropod clades were secondarily adapted to piscivory, omnivory and herbivory, and theropods show a large array of skull and dentition morphologies. This work aims to investigate aspects of the evolution of theropod dinosaurs by analyzing in detail both the anatomy and ontogeny of teeth and quadrates in non-avian theropods, and by studying embryonic and adult material of a new species of theropod. A standardized list of terms and notations for each anatomical entity of the tooth, quadrate, and maxilla is here proposed with the goal of facilitating descriptions of these important cranial and dental elements. The distribution of thirty dental characters among 113 theropod taxa is investigated, and a list of diagnostic dental characters is proposed. As an example, four isolated theropod teeth from the Lourinhã Formation (Kimmeridgian‒Tithonian) of Portugal are described and identified based on a cladistic analysis performed on a data matrix of 141 dentition-based characters coded in 60 taxa. Two shed teeth are referred to an abelisaurid, providing the first record of Abelisauridae in the Jurassic of Laurasia and the one of the oldest records of this clade in the world, suggesting a possible radiation of Abelisauridae in Europe well before the Upper Cretaceous. The consensus tree resulting from this phylogenetic analysis, the most extensive on theropod teeth, indicates that theropod teeth provide reliable data for identification at approximately family level, and this method will help identifying theropod teeth with more confidence. A detailed description of the dentition of Megalosauridae is also provided, and a discriminant analysis performed on a dataset of numerical data collected on the teeth of 62 theropod taxa reveals that megalosaurid teeth are hardly distinguishable from other theropod clades with ziphodont dentition. This study highlights the importance of detailing anatomical descriptions and providing additional morphometric data on teeth with the purpose of helping to identify isolated theropod teeth. In order to evaluate the phylogenetic potential and investigate the evolutionary transformations of the quadrate, a phylogenetic morphometric analysis as well as a cladistic analysis using 98 discrete quadrate related characters were conducted. The quadrate morphology by its own provides a wealth of data with strong phylogenetic signal, and the phylogenetic morphometric analysis reveals two main morphotypes of the mandibular articulation of the quadrate linked to function. As an example, six isolated quadrates from the Kem Kem beds (Cenomanian) of Morocco are determined to be from juvenile and adult individuals of Spinosaurinae based on phylogenetic, morphometric, and phylogenetic morphometric analyses. Morphofunctional analysis of the spinosaurid mandibular articulation has shown that the posterior parts of the two mandibular rami displaced laterally when the jaw was depressed due to a mediolaterally oriented intercondylar sulcus of the quadrate. Such lateral movement of the mandibular ramus was possible due to a movable mandibular symphysis in spinosaurids, allowing the pharynx to be widened. A new species of theropod from the Lourinhã Formation of Portugal, Torvosaurus gurneyi, is erected based on a right maxilla and an incomplete caudal centrum. This taxon supports the mechanism of vicariance that occurred in the Iberian Meseta during the Late Jurassic when the proto-Atlantic was already well formed. A theropod clutch containing several crushed eggs and embryonic material is also assigned to this new species of Torvosaurus. Investigation on the maxilla ontogeny in basal tetanurans reveals that crown denticles, elongation of the anterior ramus, and fusion of interdental plates appear at a posthatchling stage. On the other hand, maxillary pneumaticity is already present at an embryonic stage in non-avian theropods.

Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.