Matthew C. Allender

Eastern box turtles ( Terrapene carolina carolina ) are increasingly targeted by the illegal wildlife trade, which facilitates the spread of infectious diseases and complicates conservation efforts. Managing confiscated turtles, which are often positive for multiple pathogens, requires evidence‐based quarantine protocols, yet how housing conditions influence pathogen dynamics and detection remains poorly understood. We evaluated how housing configuration and temperature affect pathogen prevalence, clinical disease, and detection, and identified sampling strategies that optimize pathogen detection during quarantine. Thirteen confiscated eastern box turtles were housed in 4 environmental conditions: 1) group‐housed at cold temperatures, 2) group‐housed within a temperature gradient, 3) group‐housed at warm temperatures, and 4) individually housed at warm temperatures. We sampled turtles weekly over 8‐week trials for Terrapene herpesvirus 1 (TerHV1), Terrapene adenovirus (TerAdv), and box turtle Mycoplasmopsis sp. (BTMyco). A sensitivity analysis identified the optimal sampling approach for each condition. Apparent prevalence, clinical signs ( n = 7), and mortality ( n = 2) were highest in group‐housed turtles at warm temperatures, where BTMyco and TerAdv were detected in all turtles, and TerHV1 was detected in all but 2 turtles; however, detection and co‐detection were highest for BTMyco and TerAdv when turtles were housed within a temperature gradient. Two weekly swabs maximized detection, while sampling every 3 weeks remained effective for quarantine periods exceeding 2 weeks. These results provide practical, evidence‐based recommendations for quarantine design that reduce disease transmission risk while optimizing pathogen detection in confiscated turtle management and highlight how temperature‐driven changes in pathogen dynamics may have implications under a changing climate. Future work should validate these protocols across larger cohorts and field‐based confiscation settings and further examine how warming temperatures reshape disease risk in chelonian conservation.

Exposure to SARS-CoV-2 has been documented in a broad range of animal species, raising concerns regarding transmission within wildlife populations and their potential role as viral reservoirs for secondary zoonotic infections. Although serological surveillance of SARS-CoV-2 in animals has increased, it remains far less extensive than studies conducted in humans. Validated serological assays may serve as important tools for detecting prior SARS-CoV-2 exposure in animals. In this study, we conducted multi-species serological surveillance to assess prior exposure to SARS-CoV-2 in free-ranging wild animal populations including white-tailed deer, raccoons, fur seals, sea lions, penguins, and cormorants. A total of 589 serum samples, including samples from white-tailed deer (n = 245), raccoons (n = 147), fur seals (n = 66), sea lions (n = 14), penguins (n = 72) and cormorants (n = 45), were collected between 2022 and 2024. Samples were tested using two serological assays: an enzyme-linked immunosorbent assay targeting antibodies against the nucleocapsid protein (N-ELISA) and a surrogate virus neutralization test (sVNT) targeting antibodies against the receptor-binding domain (RBD) of the spike (S) protein. Seropositivity was determined based on established assay-specific cutoff values. Among white-tailed deer, 46 of 245 samples (18.8%) were seropositive. Of these, 38 samples were positive only by the sVNT, 7 only by N-ELISA, and only 1 sample was positive by both assays. In raccoons, 20 of 147 samples (13.6%) were positive by the sVNT and no samples were positive by N-ELISA. In contrast, among marine mammals and avian species, 3 of 197 samples (1.5%) were positive by N-ELISA, including 1 fur seal and 2 penguins, while no samples were positive by the sVNT. Overall, these findings highlight species-specific differences in SARS-CoV-2 exposure and underscore the importance of multi-species serological surveillance to better understand SARS-CoV-2 circulation, persistence, transmission, and the potential role of wildlife as reservoirs.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused a global public health emergency in humans from 2020 to 2023 and was associated with over 7 million human deaths. Besides humans, SARS-CoV-2 has been detected in a wide range of animals, including companion, farm, zoo, and wild animals. At least 61 animal species from 29 animal families of 12 animal orders have tested SARS-CoV-2 positive. Documented evidence reported that not only human-to-animal transmission but also animal-to-human transmission events occurred. During the course of the pandemic progression in humans, SARS-CoV-2 strains in animals evolved in parallel with those in humans. Continued monitoring of SARS-CoV-2 in animals is needed to safeguard both human and animal health. In this study, we report investigation of two outbreaks of SARS-CoV-2 Omicron variant infection in tigers and gorillas in two zoological institutions. In the first zoo, six tigers tested positive by SARS-CoV-2 real-time RT-PCR and shed viral nucleic acid in feces for up to two weeks. Three of the tigers showed intermittent shedding patterns, while the other tigers shed only for 7–10 days. No other species, including cheetah, otter, lion, anteater, gibbon, and tamarin, tested positive. During the outbreak at the second zoo, a total of six gorillas were tested positive for SARS-CoV-2, while other primates housed in the same building (colobus and orangutan) tested negative. Follow-up testing revealed that two gorillas tested positive for SARS-CoV-2 over a one-month period (30 and 33 days, respectively), while the other four gorillas had positive SARS-CoV-2 PCR results for 14 to 25 days. Four gorillas had intermittent shedding patterns. Notably, compared to tigers, gorillas had a prolonged duration of fecal viral shedding. Sequencing was performed on the positive samples, and analysis indicated that strains detected in tigers and gorillas belonged to SARS-CoV-2 Omicron BQ.1.10 and XBB.1.16, respectively. Overall, this study offers valuable insights into the duration of viral RNA shedding for SARS-CoV-2 Omicron in zoo animals, facilitating accurate diagnostic evaluation and management of infected tigers and gorillas.

Since its discovery in 2019, SARS-CoV-2 has continued to be detected in both humans and animals worldwide. Currently there is limited research focusing on serological surveillance of wildlife under human care. Here we tested 230 serum samples of 134 animals from two zoological institutions collected between 2015 and 2024. To assess prior exposure and antibody responses from natural infection or vaccination, we used three serological assays: a nucleocapsid protein-based ELISA (N-ELISA), a surrogate virus neutralization test (sVNT) for spike (S) protein and a neutralization assay with S-pseudotyped viral particles. Among the 114 samples collected from 58 animals at Fort Wayne Zoo in Indiana, 37 samples from 20 vaccinated animals were sVNT-positive, and 2 of the positive animals had 2 samples prior to vaccination that tested positive by N-ELISA. Of the 116 samples from 76 animals at Brookfield Zoo in Illinois, 20 samples of 20 animals were sVNT-positive, and 19 of the positive animals had been vaccinated. Among these 20 sVNT-positive samples, only one sample from a South American Tapir was positive from prior to vaccination and 1 sample from a sloth bear was also positive by N-ELISA, marking the first documented cases of SARS-CoV-2 exposure in both species. Neutralization assays with S-pseudotyped virus revealed that some of the sVNT-positive samples have strong activity against the WH1-S pseudovirus but showed significantly reduced neutralization against the Omicron LP.8.1-S pseudovirus. These results underscore the need for updated vaccines tailored to emerging variants. Overall, our findings highlight the importance of continued serological surveillance across multiple species to detect new SARS-CoV-2 exposures and monitor vaccine-induced immunity in captive animal populations.

The wide host range, potential lethality, and zoonotic potential of SARS-CoV-2 infection in animals highlights the need for additional surveillance strategies. We validated a commercial, pH-based, colorimetric RT-LAMP assay for the detection of SARS-CoV-2 RNA in animal feces. The comparator assay was rRT-PCR. The limit of detection of the RT-LAMP assay was 72 genome copies per reaction. RT-LAMP was highly specific for SARS-CoV-2 and did not detect other human or animal coronaviruses. RT-LAMP was robust, with valid results generated for incubation lengths of 30 to 45 min, incubation temperatures of 60 to 70 °C, and reaction volumes of 10 to 25 µL. The diagnostic sensitivity was 100% for clinical fecal samples with high viral loads (Ct ≤ 25), 97.4% for samples with moderate to high viral loads (Ct ≤ 33), and 62% overall (Ct ≤ 40). The diagnostic specificity was 97.9%. Blinded method testing organized by an independent laboratory confirmed the satisfactory reproducibility of the assay. To our knowledge, this study represents the first validation of RT-LAMP for SARS-CoV-2 detection in animals. RT-LAMP testing could detect SARS-CoV-2 infection more rapidly and at the point of care in animals with moderate to high viral loads, allowing for earlier implementation of control measures.

Morbidity and mortality associated with adenoviruses (AdVs) have been reported in multiple taxa, including chelonians. Adenoviruses of a distinct testudinoid origin have been detected in both managed and free-ranging turtles in North America and Europe; however, the prevalence and impact of AdVs in free-ranging turtle populations have yet to be determined. A multispecies investigation to detect novel or existing AdVs in Blanding's turtles (Emydoidea blandingii; n=1,359), painted turtles (Chrysemys picta; n=270), and red-eared sliders (Trachemys scripta elegans; n=205) was performed across four counties in Illinois (with one site reaching into Wisconsin), USA, from 2016 to 2022. Sequence-confirmed AdVs were detected in Blanding's turtles (1.8%; n=24), painted turtles (13.0%; n=35), and red-eared sliders (8.8%; n=18). Ten AdVs, including 5 novel AdVs, were detected across the three species, with each species having 3–6 AdVs detected. Three AdVs were probably prey related, whereas the remaining seven AdVs were probably host-adapted AdVs, based on the absence of clinical signs of disease. The majority of AdVs detected were testadenoviruses; however, the siadenovirus Sulawesi tortoise adenovirus (STADV) was sequence confirmed in 1 Blanding's turtle, 15 painted turtles, and 10 red-eared sliders, all of which were free of clinical signs of disease. These results provide a baseline for the presence and diversity of AdVs in free-ranging turtles in Illinois, including evidence for a North American reservoir for STADV.

During the COVID-19 pandemic, veterinary diagnostic laboratories tested both human and animal samples and needed to ensure that they could accurately perform large numbers of diagnostic tests in a timely manner. Sample pooling, a methodology used effectively for over 80 years as a surveillance tool for screening large numbers of potentially infected individuals, was employed. Given its sensitivity, real-time polymerase chain reaction (PCR) is more suitable for employing this strategy, as compared to other less sensitive testing methods. In this study, we evaluated the capability of detecting SARS-CoV-2 in both 5-sample and 10-sample pools of feces using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) as well as determined the level of sensitivity. A blinded method test (BMT) by an independent laboratory was conducted to assess the five-sample fecal pool. To complement detection capability, the stability of the genome within a PBS fecal suspension was measured under various time and temperature conditions across a 28-day period. Our results showed that the limit of detection for 5-sample and 10-sample fecal pools is 12.8 and 6.4 genome copies in a 25 µL PCR, respectively. The 5-sample and 10-sample pooling resulted in a cycle threshold (Ct) value loss of 2.35 and 3.45, as compared to Ct values of known positive individual samples, but consistent detection was still achieved in pools containing positive samples with an original Ct below 36 and 34, respectively. The simulation of clinical five-sample pooling showed that all positive samples could be detected regardless of the number (1–3) of positive samples in each pool. The BMT results demonstrated excellent sensitivity (100 copies/reaction) in five-sample pools for the detection of SARS-CoV-2 RNA even though a fecal matrix effect was observed. Finally, our results show that the SARS-CoV-2 genome remains stable over a wide range of time and temperature variations. Overall, our findings provide solid data to scale up SARS-CoV-2 testing capacity in veterinary diagnostic laboratories.