Vetnuus | September 2025 7 Leading Article >>>8 into human exposures to the disease. However, there is limited information on rabies surveillance data in the southeastern United States. This study analysed animal rabies cases submitted to the Athens Veterinary Diagnostic Laboratory (AVDL, University of Georgia, Athens, GA, USA) from 2019 to 2023. We focused on identifying any patterns in rabies cases, such as the occurrence among wild and domestic species, analysing seasonal trends, and mapping the geographical distribution of positive cases. Our findings provide useful insights for long-term policy decisions and improving rabies prevention and control strategies. MATERIALS AND METHODS Data collection and analysis We queried the Athens Veterinary Diagnostic Laboratory (AVDL) database using the Laboratory Information Management System called VetView to retrieve 1,560 rabiessuspect cases submitted to the laboratory from 2019 to 2023. Tissue samples were submitted by various clients from within and outside Georgia, including the Southeastern Cooperative Wildlife Disease Study (SCWDS). Those specimens were tested using the DFAT, and the data were collected in an Excel spreadsheet. Each case was individually reviewed to capture details, including accession number, received date, specimen type, species, geographic locations, seasonality, and diagnostic outcomes. Cases were categorised as positive and negative based on DFAT results. We categorised the positive cases by species, and seasonal trends were analysed by grouping the data into four seasons (winter, spring, summer, and fall). We examined temporal trends over the 5-year period to identify significant patterns in rabies occurrence. Positive cases were mapped for geographic distribution and further classified into cases originating from wildlife and domestic animals for comparative analysis. Direct fluorescent antibody test According to the World Health Organisation (WHO), the DFAT is considered the gold standard for rabies testing, designed to detect the presence of rabies virus (RABV) antigens in brain tissue (26). The rabies testing procedure was performed following the Centres for Disease Control and Prevention guidelines and standards (27). Brain tissue samples were collected and sectioned to include identifiable areas of the right and left lateral lobes of the cerebellum, the vermis, and the brainstem. Tissue impressions were prepared on clean glass microscope slides. These slides were then air-dried and fixed in cold acetone (−20°C) for at least an hour to preserve antigen integrity. After the fixation period, the slides were stained with three separate conjugates, including the EMD Millipore Corporation 5100 Light Diagnostics Rabies DFA Reagent (EMD Millipore Corp, Temecula, CA), Fujirebio Diagnostics Inc FITC Anti-Rabies Monoclonal Globulin (FDI, Malvern, PA), and the Millipore Light Diagnostics Rabies Negative Control, Monoclonal Antibody FITC Conjugate 5102 (EMD Millipore Corp, Temecula, CA) conjugates. Stained slides were incubated in the humid chamber for 30 min, allowing sufficient time for antibodyantigen binding. After incubation, slides were rinsed with rabbit phosphate-buffered saline (PBS) to remove unbound antibodies and examined under a fluorescent microscope’s FITC filter. The interpretation of slides was based on fluorescence intensity and antigen distribution. Positive rabies impression smears showed a bright apple-green fluorescence in rabies virus-infected neuronal cells represented by massive intracytoplasmic inclusions of various shapes (dust-like particles, large, round to oval). In all observed fields, samples considered negative displayed no fluorescence and no inclusions, and the tissue appeared as a dull red background. Before any testing, conjugates were subjected to an initial titration to determine the optimal working dilution for routine use. We prepared serial dilutions of conjugates that will be tested with control material from naturally infected animals. Brain tissues used were from previously submitted accessions, particularly a raccoon strain that was tested and confirmed rabies-positive to ensure the reliability of the results. Statistical analysis The statistical analysis was conducted using JMP Pro version software. χ2 tests were used to evaluate significant associations between the species, seasonality, and rabies occurrence, with significance determined at a P-value < 0.05. RESULTS A total of 1,560 cases were submitted for rabies testing from 2019 to 2023. Out of 1,560 cases, 94.2% [1,470/1,560] were negative for rabies, 5.6% [88/1,560] were positive for rabies across 11 species, and 0.1% [2/1,560] were non-conclusive cases. The occurrence of rabies varied between wildlife (83% [73/88]) and domestic (17% [15/88]) animals, and most rabies-positive cases were coming from Georgia. Over the 5 years, the negative and the total number of rabies submitted cases remained relatively stable until 2021, when there was a decrease followed by an increasing trend starting in 2022. The number of positives also remained stable, with a slight peak in 2022 (Fig. 1b). Wildlife species significantly accounted for most of the positive cases, with 83% (73/88) representing seven species, whereas 17% (15/88) were from domestic animals (P-value < 0.0001) (Fig. 1b). As shown in Fig. 1c, among wildlife, the affected species were raccoons (35.2% [31/88), skunks (25% [22/88]), white-tailed deer (8% [7/88]), foxes (6.8% [6/88]), bats (4.5% [4/88]), bobcats (2.3% [2/88]), and great kudu (1.1% [1/88]). In domestic animals, the affected species were bovine (6.8% [6/88]), feline (5.7% [5/88]), caprine (2.3% [2/88]), and equine (2.3% [2/88]) (Fig. 1d). The positivity rates also differ by species. Skunks (38.6% [22/57]) had the highest positivity rate among wildlife species, followed by great kudu (20% [1/5]), raccoons (12.3% [31/253]), foxes (10.7% [6/56]), bobcats (8.3% [2/24]), bats (7.8% [4/51]), and white-tailed deer (6.5% [7/107]). The domestic species, including bovine (13.3% [6/45]), equine (8.3% [2/24]), caprine (4.3% [2/47]), and feline (4.1% [5/121]), also displayed a difference in positivity rates. Figure 2 shows the distribution of rabies cases across most Southeastern states and Washington, D.C. Positive cases were predominantly located in Georgia (88.6% [78/88]), with additional cases identified in Washington, D.C. (2.3% [2/88]), Louisiana (1.1% [1/88]), and South Carolina (1.1% [1/88]). The remaining positive cases were reported with unknown locations (6.8% [6/88]). Additionally, we observed a seasonal variation in positive cases throughout the years, with noticeable peaks occurring during certain months. In 2023, specifically, the results showed a very sharp peak characteristic of an increase in positive cases from July until October (Fig. 3). The infection rates during fall (6.9% [26/379]), spring (6% [23/382]), and summer (5.7% [26/456]) were higher than in winter (3.8% [13/343]). The highest proportion of submitted cases was observed during the summer (29.2% [456/1,560]), and 24.3% [379/1,560], 24.5% [382/1,560], and 22% [343/1,560] accounted for submissions received during the fall, spring, and winter, respectively. There is, however, no significant statistical association between seasons and the occurrence of rabies cases (P-value = 0.3387).
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