Denise Pesti

Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization

The evaluation of the usefulness of DNA probes in a diagnostic setting to identify nuclear inclusions in selected viral infections (psittacine beak and feather disease viral infection, avian polyomavirus infection, adenovirus infection and Pachecos parrot disease) is reported. A DNA in situ hybridization method was used to detect viral nucleic acid in sections of paraffin-embedded tissues coming from birds naturally and/or experimentally infected. It is concluded that DNA probes used for polyomavirus (FN-19) and adenovirus (FN-23) are able to identify nucleic acid of each virus in the cells with nuclear inclusions, and when used for psittacine beak and feather disease virus (FN-8), and Pachecos parrot disease virus (FN-49) are able to detect viral nucleic acid in cells with or without inclusions.

Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes

Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer-membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer-membrane proteins (Pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the Pmps and tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease.

Production and Characterization of Monoclonal Antibodies to Psittacine Beak and Feather Disease Virus

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

Atypical chlamydiaceae in wild populations of hawks (Buteo spp.) in California

Abstract Chlamydiaceae bacteria infect many vertebrate hosts, and previous reports based on polymerase chain reaction (PCR) assays and serologic assays that are prone to cross-reaction among chlamydial organisms have been used to describe the prevalence of either DNA fragments or antibodies to Chlamydia spp. in wild raptorial populations. This study reports the PCR-based prevalence of Chlamydiaceae DNA that does not 100% match any avian or mammalian Chlamydiaceae in wild populations of hawks in California Buteo species. In this study, multimucosal swab samples (n = 291) for quantitative PCR (qPCR) and plasma (n = 78) for serology were collected from wild hawks. All available plasma samples were negative for antibodies using a C. psittaci–specific elementary body agglutination test (EBA; n = 78). For IgY antibodies all 51 available samples were negative using the indirect immunofluorescent assay. The overall prevalence of Chlamydiaceae DNA detection in wild Buteo species sampled was 1.37% (4/291) via qPCR-based analysis. Two fledgling Swainsons hawks (Buteo swainsoni) and two juvenile red-tailed hawks (Buteo jamaicensis) were positive by qPCR-based assay for an atypical chlamydial sequence that did not 100% match any known C. psittaci genotype. Positive swab samples from these four birds were sequenced based on the ompA gene and compared by high-resolution melt analysis with all known avian and mammalian Chlamydiaceae. The amplicon sequence did not 100% match any known avian chlamydial sequence; however, it was most similar (98.6%) to C. psittaci M56, a genotype that is typically found in muskrats and hares. Culture and full genome sequence analysis of Chlamydia spp. isolated from diseased hawks will be necessary to classify this organism and to better understand its epizootiology and potential health impact on wild Buteo populations in California.

CHLAMYDIA PSITTACI IN FERAL ROSY-FACED LOVEBIRDS (AGAPORNIS ROSEICOLLIS) AND OTHER BACKYARD BIRDS IN MARICOPA COUNTY, ARIZONA, USA

Abstract:  In 2013, a mortality event of nonnative, feral Rosy-faced Lovebirds (Agapornis roseicollis) in residential backyards in Maricopa County, Arizona, US was attributed to infection with Chlamydia psittaci. In June 2014, additional mortality occurred in the same region. Accordingly, in August 2014 we sampled live lovebirds and sympatric bird species visiting backyard bird feeders to determine the prevalence of DNA and the seroprevalence of antibodies to C. psittaci using real-time PCR-based testing and elementary body agglutination, respectively. Chlamydia psittaci DNA was present in conjunctival-choanal or cloacal swabs in 93% (43/46) of lovebirds and 10% (14/142) of sympatric birds. Antibodies to C. psittaci were detected in 76% (31/41) of lovebirds and 7% (7/102) of sympatric birds. Among the sympatric birds, Rock Doves (Columba livia) had the highest prevalence of C. psittaci DNA (75%; 6/8) and seroprevalence (25%; 2/8). Psittacine circovirus 1 DNA was also identified, using real-time PCR-based testing, from the same swab samples in 69% (11/16) of species sampled, with a prevalence of 80% (37/46) in lovebirds and 27% (38/142) in sympatric species. The presence of either Rosy-faced Lovebirds or Rock Doves at residential bird feeders may be cause for concern for epizootic and zoonotic transmission of C. psittaci in this region.