Honors Theses and Capstones

Date of Award

Spring 2023

Project Type

Senior Honors Thesis

College or School

COLSA

Department

Molecular, Cellular, and Biomedical Sciences

Program or Major

Biomedical Science

Degree Name

Bachelor of Science

First Advisor

David B. Needle

Abstract

From May through July 2021, an unusual mortality event occurred along the eastern coast and Midwest of the United States. Thousands of birds, mostly from the order Passeriformes, were part of the die-off including blue jays (Cyanocitta cristata), common grackles (Quiscalus quiscula), European starlings (Sturnus vulgaris), American robins (Turdus migratorius). Clinical signs included crusted eyes, swollen conjunctiva, otitis, seizures, and ataxia.

The New Hampshire Veterinary Diagnostic Laboratory (NHVDL) received over 100 affected birds from various collaborators throughout the United States including Washington DC, NJ, CT, MD, and OH. Given the timing and geologic distribution of the deaths, several hypotheses were being investigated including the possibility that a virus contracted during migration was spreading, or that disease was being passed through the food chain from excessive insecticide use or the emerging Brood X Cicadas.

Prior to submission, all birds were frozen and shipped via a next-day courier service. Once received at the NHVDL, they were thawed for tissues to be collected. A portion of conjunctiva, ear canal tissue, lung, liver, and feces was collected from each specimen received. DNA was extracted from the samples using the MagMax DNA Multi-Sample Ultra 2.0 kit. Library preparation was performed using the Kapa HyperPrep kit with TruSeq adapters and sequencing was completed on an Illumina NovaSeq 6000 instrument which produced 250 bp paired-end reads for two different datasets. The datasets were first compared to the most closely related reference genome to filter out avian reads. Then, the unmapped reads were assembled following a standard whole genome sequencing workflow.

There were many bacterial and viral genomes identified in each sample, but overall, there was no single common pathogen in all samples. Of the viral species identified within the first batch of extractions, an average of 8.46% of the genome was labeled undefined or unresolved. Most identified viruses only appeared in one sample. Gallid alphaherpesvirus 2, Murine leukemia virus-related retrovirus, Psittacid alphaherpesvirus 1, and Xestia c-nigrum granulovirus were all found in two of the first 12 samples. Additionally, Pseudaletia unipuncta granulovirus was found in four samples. Similar to the viral findings, an average of 3.25% of the bacterial findings were labeled as undefined or unresolved. Of the thousands of identified bacterial species, they belonged to seven different orders.

The finding of Gallid alphaherpesvirus 2 is unusual as this virus is more commonly known as Merick’s disease which is ubiquitous with the ownership of chickens and is spread through infected feather dander. The presence of Murine leukemia virus-related retrovirus may also be an incidental finding because similar viruses have been identified in chickens (Borysenko et al.). Psittacid aplhaherpesvirus 1 was of interest because this is the causative agent of Pacheco’s disease, a highly contagious respiratory illness in parrots. Xestia c-nigrum granulovirus and Pseudaletia unipuncta granulovirus are both viruses that infect insects. Xestia c-nigrum is more commonly known as the Lesser Black-letter Dart, and Pseudaletia unipuncta is commonly known as the Armyworm moth. Both species of moth are found throughout North America. Furthermore, both viruses relating to insects were Granuloviruses which have been used for pest management strategies (Sood et al. 2019).

The results of this study did not reveal a conclusive explanation for the high mortality rate of songbirds observed in 2021. The next steps of this study would be to analyze the other samples at a deeper level and compare the viral taxonomic composition found in their genome. Additionally, these results have the potential to give rise to more research such as investigating the ecological impacts of each virus. A follow-up study on the Psittacid herpesvirus is currently underway with samples from birds in New Jersey.

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