Cryptosporidium infection in ostriches (Struthio camelus) in Brazil: clinical, morphological and molecular studies

Avian cryptosporidiosis has been reported in more than 30 species of birds. To date, the species infecting birds are C. baileyi, C. galli and C. meleagridis. In this study, the morphological, clinical and molecular characteristics of a Brazilian ostrich isolate of Cryptosporidium are described. The oocysts of this Brazilian isolate are larger and more elongated than those of Cryptosporidium previously reported in ostriches, which were morphologically similar to C. meleagridis. Morphological, biological and molecular analyses demonstrated similarity of this ostrich isolate with C. baileyi, suggesting that there are at least two Cryptosporidium species infecting ostriches; one with molecular, biological and morphological characteristics related to C. baileyi, and another morphologically similar to C. meleagridis

Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells

Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. In this study, we designed specific short interfering RNA (siRNA) targeting the AMPV nucleoprotein (N) and fusion (F) genes. Three days post-virus infection, virus titration, real time RT-PCR, and RT-PCR assays were performed to verify the effect of siRNA in AMPV replication. A marked decrease in virus titers from transfected CER cells treated with siRNA/N was observed. Also, the production of N, F, and G mRNAs in AMPV was decreased. Results indicate that N-specific siRNA can inhibit virus replication. In future studies, a combination of siRNAs targeting the RNA polymerase complex may be used as a tool to study AMPV replication and/or antiviral therapy. (c) 2006 Elsevier B.V. All rights reserved.741778

Genetic Diversity of Avian Infectious Bronchitis Virus Isolated from Domestic Chicken Flocks and Coronaviruses from Feral Pigeons in Brazil Between 2003 and 2009

To detect the presence of infectious bronchitis virus or avian coronavirus, a nested reverse transcriptase PCR (RT-PCR) method was developed with the aim of amplifying a fragment of 530 bases, comprising the gene coding SI protein. In the first step, all samples were submitted to RNA extraction, RT-PCR, and nested PCR. Next, only the positive nested-PCR samples were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for virus isolation. Positive samples were then sequenced and analyzed using a molecular phylogeny approach. Tracheal swab samples were collected from 23 different domestic chickens distributed in three regions of Brazil, in the period between 2003 and 2009. Also analyzed were six swab samples (tracheal and cloacal) from asymptomatic pigeons (Columba livia), caught in an urbanized region in southeastern Brazil. The study revealed two major phylogenetic groups: one clustered with the Massachusetts vaccine serotype and another joined with the D207 strain. Interestingly, samples grouped with the Connecticut and Arkansas serotypes were also found. Pigeon isolates clustered with the Massachusetts serotype showed significant similarity (close to 100%) to those obtained from chickens. Only one pigeon isolate was seen to be grouped with the Connecticut serotype, and no correlation was observed between sample grouping and region origin. Understanding the diversity of genotypes and eco-epizootiology of the disease in different environments is expected to be helpful for vaccine production aimed at the main circulating variants. In this respect, one could also expect benefits in the management of other bird species that may act as avian coronavirus reservoirs.5441191119

Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F-based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10(1) TCID(50) mL(-1)). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N-and F-based RRT-PCR and they can be successfully used for AMPV/A detection.39514451451Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)[03 14012-9]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)[03 14012-9

Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, Sao Paulo

Rosa G.N., Domingues H.G., Santos M.M.A.B., Felippe P.A.N., Spilki F.R. & Arns C. W. 2012. [Molecular detection and phylogenetic analysis of the gene H from canine distemper virus isolates circulating at the municipality of Campinas, Sao Paulo.] Deteccao molecular e analise filogenetica do gene H de amostras do virus da cinomose canina em circulacao no municipio de Campinas, Sao Paulo. Pesquisa Veterinaria Brasileira 32(1): 72-77. Laboratorio de Microbiologia Molecular, Instituto de Ciencias da Sa de, Universidade Feevale, Rodovia RS-239 2755, Novo Hamburgo, RS 93352-000, Brazil. E-mail: [email protected] Canine distemper virus (CDV), a Morbillivirus of the family Paramyxoviridae, is the etiological agent of neurological and systemic disease in dogs. The laboratory diagnosis of infection requires viral isolation or detection of genetic material of the virus in secretions or tissues of dogs with clinical suspicion of the disease. The genetic diversity among isolates of CDV can be assessed by sequencing and phylogenetic analysis of the gene that encodes the viral hemagglutinin (H gene), and there is currently a special interest in comparing the strains currently circulating in the field with the genogroup America-1, which comprises strains present in vaccines available in the market. In this study, the molecular detection of CDV gene H was performed from biological samples harvested ante-and post-mortem from 15 dogs with clinical signs suggestive of canine distemper in the metropolitan region of Campinas, Sao Paulo. Ten of the 15 dogs examined had at least one positive organ under molecular detection and the obtained amplicons were sequenced and further analyzed by molecular phylogenetic analysis. Similarly to what has already been reported on previous studies regarding the diversity of the gene H in other countries, the phylogenetic reconstruction obtained for the samples of cases of distemper from Campinas region showed they were grouped with the North American, European and Japanese newly described samples, a genetic group distinguished from classical samples of CDV, named America-1, which encompasses the vaccine strains Snyder Hill, Onderstepoort and Lederle..321727