A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

BACKGROUND: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. METHODS: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. RESULTS: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. CONCLUSIONS: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Characterization of Avian Pox in a Ruffed Grouse (Bonasa umbellus) from Pennsylvania.

A free-ranging, adult male ruffed grouse (Bonasa umbellus) was harvested by a hunter during November 2019 in Forest County, PA. The bird was submitted for necropsy due to a skin mass on its left leg. Upon necropsy, two proliferative skin masses were grossly visible, one on the left leg and one on the cere. An additional mass was present on the oropharyngeal mucosa covering the hard palate. These masses were diagnosed as avian pox based on histopathologic and cytologic findings, including marked epithelial hypertrophy, hyperplasia, vacuolar degeneration with eosinophilic stippling, and intracytoplasmic inclusion bodies. An avipoxvirus was detected using PCR and was identified as fowlpox virus through sequencing of the 4b core gene segment. The avipoxvirus from this case showed genetic similarity to isolates from Eastern wild turkeys (Meleagris gallopavo silvestris).

Caracterización de la viruela aviar en un grévol engolado (Bonasa umbellus) en el estado de Pensilvania. Un cazador recolectó un grévol engolado macho adulto silvestre (Bonasa umbellus) durante noviembre del 2019 en el condado de Forest, Pensilvania. El ave fue sometida a necropsia debido a una masa cutánea en su pata izquierda. Durante la necropsia, dos masas cutáneas proliferativas fueron claramente visibles, una en la pierna izquierda y otra en la cera. Había una masa adicional en la mucosa orofaríngea que cubría el paladar duro. Estas masas se diagnosticaron como viruela aviar con base en los hallazgos histopatológicos y citológicos, que incluyeron hipertrofia epitelial marcada, hiperplasia, degeneración vacuolar con punteado eosinofílico y cuerpos de inclusión intracitoplasmáticos. Se detectó un avipoxvirus mediante PCR y se identificó como virus de la viruela aviar mediante la secuenciación del segmento del gene 4b del centro viral. El avipoxvirus de este caso mostró similitud genética con aislamientos de pavos salvajes del este (Meleagris gallopavo silvestris).