Chlamydia buteonis in birds of prey presented to California wildlife rehabilitation facilities.

Chlamydial infections, caused by a group of obligate, intracellular, gram-negative bacteria, have health implications for animals and humans. Due to their highly infectious nature and zoonotic potential, staff at wildlife rehabilitation centers should be educated on the clinical manifestations, prevalence, and risk factors associated with Chlamydia spp. infections in raptors. The objectives of this study were to document the prevalence of chlamydial DNA shedding and anti-chlamydial antibodies in raptors admitted to five wildlife rehabilitation centers in California over a one-year period. Chlamydial prevalence was estimated in raptors for each center and potential risk factors associated with infection were evaluated, including location, species, season, and age class. Plasma samples and conjunctiva/choana/cloaca swabs were collected for serology and qPCR from a subset of 263 birds of prey, representing 18 species. Serologic assays identified both anti-C. buteonis IgM and anti-chlamydial IgY antibodies. Chlamydial DNA and anti-chlamydial antibodies were detected in 4.18% (11/263) and 3.14% (6/191) of patients, respectively. Chamydial DNA was identified in raptors from the families Accipitridae and Strigidae while anti-C.buteonis IgM was identified in birds identified in Accipitridae, Falconidae, Strigidae, and Cathartidae. Two of the chlamydial DNA positive birds (one Swainson's hawk (Buteo swainsoni) and one red-tailed hawk (Buteo jamaicensis)) were necropsied, and tissues were collected for culture. Sequencing of the cultured elementary bodies revealed a chlamydial DNA sequence with 99.97% average nucleotide identity to the recently described Chlamydia buteonis. Spatial clusters of seropositive raptors and raptors positive for chlamydial DNA were detected in northern California. Infections were most prevalent during the winter season. Furthermore, while the proportion of raptors testing positive for chlamydial DNA was similar across age classes, seroprevalence was highest in adults. This study questions the current knowledge on C. buteonis host range and highlights the importance of further studies to evaluate the diversity and epidemiology of Chlamydia spp. infecting raptor populations.

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.