In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Widespread surveillance for SARS-CoV-2 was conducted across wildlife, captive animals in zoological collections, and domestic cats in Nebraska from 2021 to 2023. The goal of this effort was to determine the prevalence, phylogenetic and spatial distribution characteristics of circulating SARS-CoV-2 variants using various diagnostic methodologies that can utilize both antemortem and postmortem samples, which may be required for wildlife such as white-tailed deer. Statewide surveillance testing revealed high variation in SARS-CoV-2 prevalence among species, with white-tailed deer identified as the primary reservoir. In 2021, seroprevalence in white-tailed deer was 63.73% (n = 91) and 39.66% (n = 237) in 2022, while virus detection in retropharyngeal lymph nodes (RLN) was 16.35% (n = 483) in 2021 and 3.61% (n = 277) in 2022. Phylogenetic analysis was conducted on 11 positive samples from 2021. This analysis revealed the presence of four lineages of the Delta variant: AY.100, AY.119, AY.3, and AY.46.4. Conversely, other species showed no virus detection, except domestic cats, which had a low seroprevalence of 2.38% (n = 628) in 2022, indicating minimal exposure. The detection of SARS-CoV-2 in white-tailed deer and the identification of multiple Delta lineages underscores the need for ongoing surveillance and the importance of using different diagnostic methodologies. These efforts are critical for understanding virus circulation and evolution in wildlife and domestic animals, informing public health strategies, and mitigating the risks of zoonotic transmission of SARS-CoV-2 and other emerging infectious diseases.

Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real-time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4°C or 25°C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4°C for 5 days with a mean Cq 26.34 (95% CI: 23.11–29.58) and detected at −20°C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73–31.37). A significant decrease in detectable RNA was observed in samples containing <10 parasites/extraction at −20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs.

Bovine Viral Diarrhea Virus (BVDV) is a significant pathogen of cattle, causing substantial economic losses. Identifying persistently infected (PI) animals is crucial for BVD control and prevention, with pooled ear-notch tissue testing being a standard screening method. Digital PCR (dPCR) is an emerging diagnostic tool that provides absolute quantification of animal pathogens in low abundant targets. This study evaluates the effectiveness of dPCR for BVD surveillance using field submissions of pooled ear-notch samples, comparing it to real-time quantitative reverse transcription PCR (qRT-PCR). The study compared performance using 107 pools of ear notches (4853 sample animals), including 76 BVD-positive pools (3543 sample animals) and 31 BVD-negative pools (1310 sample animals). Pools included those that contained PI animals detected by IHC (n = 25) and those with only virus detection that were negative by IHC (n = 51). Performance metrics evaluated included analytical and diagnostic sensitivity and specificity, limit of detection (LOD) and test agreement with qRT-PCR. Results showed that dPCR exhibited enhanced sensitivity and lower LOD compared to qRT-PCR, detecting as low as 0.6 viral copies/μL. dPCR achieved 100 % sensitivity (76/76) and 96.77 % specificity (30/31) compared to qRT-PCR and no detection of common bovine pathogens. The comparison between qRT-PCR and dPCR using Cohen's kappa coefficient in pooled samples was 0.98, indicating almost perfect agreement. In pooled ear-notch samples with qRT-PCR quantification cycle (Cq) values ranging from 30 to 33.99, the viral load ranged from 4.75 to 194.78 viral copies/μL. The study suggests that dPCR is a sensitive and specific method for detecting BVD in pooled ear-notches.

Tritrichomonas foetus infection can cause reproductive losses and subsequent economic losses in cow-calf herds. Our objective was to determine if 0.9% sterile saline (saline) was noninferior to PBS as a transport medium for T. foetus reverse-transcription real-time PCR (RT-rtPCR) testing. Transport tubes were prepared with either 1.6 mL of PBS with preputial washing ( n = 30) or 1.6 mL of saline with preputial washing ( n = 30) and inoculated at 3 different concentrations of T. foetus organisms (high = 100 org/100 µL; moderate = 10 org/100 µL; low = 1 org/100 µL) for a total volume of 1.7 mL/tube. Samples were submitted to a diagnostic laboratory for RT-rtPCR analysis. The effect of concentration and medium on mean Ct values was tested using linear regression. Normalized means were used to conduct a noninferiority t -test, with a noninferiority margin of 1 Ct. For all analyses, α = 0.05. Mean Ct values (high = 18.5; moderate = 22.7; low = 30.4) differed by concentration ( p <0.0001), whereas medium ( p = 0.67) and the interaction of medium and concentration ( p = 0.87) were not significant. The normalized mean Ct for PBS was 22.6 (min = 16.2; max = 27.0), and for saline was 22.8 (min = 20.9; max = 26.4). The normalized mean Ct of saline was noninferior to PBS ( p = 0.037). The use of saline as a transport medium for T. foetus RT-rtPCR should not affect Ct results.

The reliability of assumptions made about prevalence for pooling optimization varies greatly among target pathogens and surveillance strategies. When prevalence is unknown and difficult to anticipate, surveillance programs risk generating additional costs if pooling is suboptimal. Different methods of approximating optimal pool size (OPS) vary in precision of optimization, required sampling information, and the logistical demands placed on a laboratory workflow. Hence, it can be unclear how to assess compatibility between pooling optimization methods and the priorities of a surveillance program, sampling practices for the target population, and infection dynamics of the target pathogen. Our aim was to determine the relative performance in maximizing testing economy and cost reduction in different surveillance programs by simulating different pooling optimization methods on data from 280 submissions for bovine viral diarrhea virus (BVDV) surveillance (Nebraska Veterinary Diagnostic Center) and 111 submissions for Theileria orientalis surveillance (Virginia Tech Animal Laboratory Services). True prevalence, OPS, and historical prevalence were determined for each submission, and different optimization methods using fixed pool sizes, historical prevalence, and prevalence estimation testing were trialed on the data through Monte Carlo simulations. Contrasting results were observed between the 2 target pathogens, with historical prevalence being the most reliable optimization method for BVDV and the least reliable method for T. orientalis , which required significantly more tests than truly optimized pooling ( p <0.05). Our results demonstrate the need to consider the interplay of infection dynamics, sampling practices, and surveillance priorities when selecting a pooling optimization approach.