Avian reovirus (ARV) has emerged as an important pathogen in turkeys, causing economic losses through tenosynovitis, necrotizing hepatitis, immunosuppression, and enteric disease. Despite its ubiquity, the evolutionary history of ARV cross-species transmission among chickens, turkeys, and wild birds remains poorly understood, hindering effective control and surveillance. This study investigates ARV temporal phylogenetics with an emphasis on interspecies transmission in turkeys. Whole genome sequences (WGSs) from seventy-seven turkey cases and one quail case at the Iowa State University Veterinary Diagnostic Laboratory, along with 74–136 segment sequences per gene from GenBank (1970–2023), were analyzed. Temporal phylogenetic analyses identified chickens as the ancestral host, with spillover into turkeys beginning in the mid-20th century, followed by stable transmission within turkey populations. Migration analyses revealed predominantly unidirectional transmission from chickens to turkeys. WGS analyses showed high variability in the M2 and σC-encoding region of the S1 segment, suggesting selective pressure on outer capsid proteins. M2, S1 σC, and L3 had the highest substitution rates, implicating their role in adaptation and antigenic diversity. These findings highlight the complexity of ARV evolution across hosts and underscore the need for robust genotyping schemes and surveillance strategies to mitigate outbreaks in poultry.

Ornithobacterium rhinotracheale (ORT) has been associated with poultry respiratory disease worldwide. The organism is fastidious and isolation is challenging. One TaqMan real-time PCR (qPCR) assay has been developed for the detection of ORT. However, during validating the ORT qPCR, the assay performance was suboptimal. During the in silico evaluation, deviations from the basic parameters for primers and probes designs (e.g., presence of stable undesirable primer-dimers) were observed. The suboptimal design led to low efficiency and low sensitivity of the assay. Initially, modification on the probe was carried out to improve the performance of the assay. However, the assay’s performance (efficiency and sensitivity) was still suboptimal. In this manuscript, we describe the development of a new qPCR assay and the comparison of its performance with the currently available assay. A highly efficient, sensitive, and specific qPCR assay was developed with approximately 1000-folds reduction in the limit of detection (from 3 × 10⁶ plasmid DNA copies/mL to 1 × 10³ plasmid DNA copies/mL). Additionally, the efficiency of the new assay (E = 98.70%) was significantly better than the current assay (E = 73.18%). The newly developed assay is an improved diagnostic tool for the sensitive and efficient diagnosis of ORT from clinical samples.

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 10³ plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.

We report the complete genome sequences of two non-typical Avibacterium paragallinarum (AP) strains isolated from chickens in the absence of clinical signs. The availability of these genomes can aid scientists in improving current diagnostics and increase our understanding of AP epidemiology and pathogenicity in chickens.

Infectious coryza (IC) caused by Avibacterium paragallinarum (AP) is an emerging infectious respiratory disease in the commercial chicken layer industry in the Midwestern US states. Outbreak investigations around positive index cases led to the discovery of nonpathogenic AP (npAP), which caused quantitative real‐time PCR (qPCR) positive results in naïve‐healthy layer (NHL) flocks. Therefore, the reliability of positive qPCR as confirmed diagnosis of IC became questionable and the poultry industry was deprived from an essential diagnostic tool in the face of an actively spreading outbreak. However, the prevalence of npAP in NHL flocks and the magnitude of this diagnostic challenge remained unclear. This pilot surveillance study aims to provide an initial estimate of npAP prevalence in the US commercial layer industry. Two differential qPCR assays were recently developed to differentiate pathogenic AP (pAP) and npAP. A total of 710 oropharyngeal (OP) swab pools (5 swabs/pool) were collected from 80 NHL sites across 13 US states and tested using qPCR assay targeting the recN gene as a screening test. Two hundred thirty‐one out of 710 total pools were positive for npAP (32.5%) representing 28 positive sites out of the 80 total sites (35%). All positive qPCR samples from NHL flocks were confirmed to be due npAP. The differential qPCR was capable of confirming 85.71% of the npAP cases, while the remaining cases required further isolation and whole‐genome sequencing (WGS). In conclusion, this pilot study indicates that the prevalence of npAP in NHL flocks in the United States is above 30%. Therefore, in flocks with no clinical signs, qPCR assays cannot be relied upon for IC diagnostic confirmation. Currently, isolation combined with WGS is the only diagnostic tool capable of completely differentiating between these two AP populations, which indicates the immediate need for improvements in the available diagnostic assays.

Avian influenza virus (AIV) is a significant threat to the poultry industry, necessitating rapid and accurate diagnosis. The current AIV diagnostic process relies on virus identification via real-time reverse transcription–polymerase chain reaction (rRT-PCR). Subsequently, the virus is further characterized using genome sequencing. This two-step diagnostic process takes days to weeks, but it can be expedited by using novel sequencing technologies. We aim to optimize and validate nucleic acid extraction as the first step to establishing Oxford Nanopore Technologies (ONT) as a rapid diagnostic tool for identifying and characterizing AIV from clinical samples. This study compared four commercially available RNA extraction protocols using AIV-known-positive clinical samples. The extracted RNA was evaluated using total RNA concentration, viral copies as measured by rRT-PCR, and purity as measured by a 260/280 absorbance ratio. After NGS testing, the number of total and influenza-specific reads and quality scores of the generated sequences were assessed. The results showed that no protocol outperformed the others on all parameters measured; however, the magnetic particle-based method was the most consistent regarding C_T value, purity, total yield, and AIV reads, and it was less error-prone. This study highlights how different RNA extraction protocols influence ONT sequencing performance.