September 2025 The Monthly Magazine of the SOUTH AFRICAN VETERINARY ASSOCIATION Die Maandblad van die SUID-AFRIKAANSE VETERINÊRE VERENIGING Gastrointestinal disorders of backyard poultry – Part 2 of 2 CPD THEME Rabies and Rhinos nuus•news Access to CPD Articles: https://www.sava.co.za/vetnews-2025/ VET
Dagboek • Diary Ongoing / Online 2025 September 2025 SAVETCON: Webinars Info: Corné Engelbrecht, SAVETCON, 071 587 2950, corne@savetcon.co.za / https://app.livestorm.co/svtsos Acupuncture – Certified Mixed Species Course Info: Chi University: https://chiu.edu/courses/cva#aboutsouthafrica@tcvm.com SAVA Johannesburg Branch CPD Events Monthly - please visit the website for more info. Venue: Johannesburg Country Club Info: Vetlink - https://savaevents.co.za/ Eastern Cape and Karoo Branch Congress 12-13 September Venue: Radisson Blu Hotel, Port Elizabeth Info: https://vetlink.co.za/eastern_cape_and_karoo_branch/ 5th International Congress on Parasites of Wildlife and 53rd Annual PARSA Conference 14-18 September Venue: Skukuza, Kruger National Park, Mpumalanga Info: corne@savetcon.co.za or visit www.savetcon.co.za October 2025 Oranje Vaal CPD Day 11 October Venue: Afridome, Parys Info: conference@savetcon.co.za Northern Natal and Midlands Branch Congress 11-12 October Venue: Fordoun Hotel and Spa, Midlands Info: https://vetlink.co.za/northern_natal_and_midlands/ The Middle East & Africa Veterinary Congress (MEAVC) 17 -19 October: Pre- and Main Congress Workshops Venue: Jafza One Convention Centre, Dubai Info: www.meavc.com SAVA Free State and Northern Cape Branch Congress 17-18 October Venue: Goose Hill Guest Farm, Bloemfontein Info: conference@savetcon.co.za KwaZulu-Natal Branch Congress 25-26 October Venue: San Lameer Resort, Southbroom Info: www.vetlink.co.za 11th International Sheep Veterinary Congress 27-31 October Venue: Wollongong, New South Wales, Australia Info: https://intsheepvetassoc.org/11th-isvc-2025 10th Annual South African Immunology Society (SAIS) Congress 30 October – 01 November Venue: Garden Court Marine Parade, Durban (KZN) Info: corne@savetcon.co.za or visit www.savetcon.co.za Southern Cape Branch Congress 31 October – 01 November Venue: Oubaai Hotel Golf & Spa, George Info: https://vetlink.co.za/southern-cape-branch/
Vetnuus | September 2025 1 Contents I Inhoud President: Dr Ziyanda Qwalela president@sava.co.za Interim Managing Director: Dr Paul van der Merwe md@sava.co.za Editor VetNews: Ms Andriette van der Merwe vetnews@sava.co.za Accounts / Bookkeeping: Ms Shaye Hughes accounts@sava.co.za/+27 (0)12 346 1150 Secretary: Ms Sonja Ludik sonja@sava.co.za/ +27 (0)12 346 1150 Reception: Ms Hanlie Swart reception@sava.co.za/ +27 (0)12 346 1150 Marketing & Communications: Ms Sonja van Rooyen marketing@sava.co.za/ +27 (0)12 346 1150 Membership Enquiries: Ms Debbie Breeze debbie@sava.co.za/ +27 (0)12 346 1150 Vaccination Booklets: Ms Debbie Breeze debbie@sava.co.za/ +27 (0)12 346 1150 South African Veterinary Foundation: Ms Debbie Breeze savf@sava.co.za/ +27 (0)12 346 1150 Community Veterinary Clinics: Ms Claudia Cloete manager@savacvc.co.za/ +27 (0)63 110 7559 SAVETCON: Ms Corné Engelbrecht corne@savetcon.co.za/ +27 (0)71 587 2950 VetNuus is die amptelike publikasie van die Suid Afrikaanse Veterinêre Vereeniging (SAVV). Alle regte word voorbehou. Geen deel van hierdie publikasie mag aangehaal, gedupliseer, versprei of aan die publiek beskikbaar gestel word in enige vorm sonder die uitdruklike skriftelike toestemming van die SAVV nie.Hierdie publikasie is uitsluitelik bedoel vir veearts en veearts verwante professionele persone soos deur die Suid Afriaanse Veterinêre Raad erken word. Wyl alles moontlik gedoen word om om die akkuraatheid van die inhoud te verseker, aanvaar nie die redaksie, SAVV of enige van die personeel, lede, werknemers of agente enige regsaanspreeklikheid vir enige verlies, skade of bevooroordeeldheid, hetsy direk of indirek, wat mag spruit uit enige stelling, feit, opinie, advertensie of aanbeveling hierin gepubliseer. Enige advertensie of verwysing na n spesifieke produk is toevallig en word nie noodwending onderskryf of aanbeveel deur die SAVV nie. VetNews is the official publication of the South African Veterinary Association (SAVA). All rights are reserved. No part of this publication may be quoted, reproduced, distributed, or made publicly available in any form or by any means without the prior express written consent of SAVA. This publication is intended solely for veterinarians and paraveterinary professionals as recognised by the South African Veterinary Council. While every effort is made to ensure the accuracy of the content, neither the editorial board, SAVA, nor any of its office bearers, members, employees, or agents shall be held liable for any loss, damage, or prejudice, whether direct or consequential, arising from any statement, fact, opinion, advertisement, or recommendation published herein. The inclusion of advertising or reference to specific products or services does not imply endorsement by SAVA. STREET ADDRESS 47 Gemsbok Ave, Monument Park, Pretoria, 0181, South Africa POSTAL ADDRESS P O Box 25033, Monument Park Pretoria, 0105, South Africa TELEPHONE +27 (0)12 346-1150 FAX General: +27 (0) 86 683 1839 Accounts: +27 (0) 86 509 2015 WEB www.sava.co.za CHANGE OF ADDRESS Please notify the SAVA by email: debbie@sava.co.za or letter: SAVA, P O Box 25033, Monument Park, Pretoria, 0105, South Africa CLASSIFIED ADVERTISEMENTS (Text to a maximum of 80 words) Sonja van Rooyen assistant@sava.co.za +27 (0)12 346 1150 DISPLAY ADVERTISEMENTS Sonja van Rooyen assistant@sava.co.za +27 (0)12 346 1150 DESIGN AND LAYOUT Sonja van Rooyen PRINTED BY Business Print: +27 (0)12 843 7638 VET Diary / Dagboek II Dagboek • Diary Regulars / Gereeld 2 From the President 4 Editor’s notes / Redakteurs notas Articles / Artikels 6 Insights into the occurrence of rabies viruses in multi-species animals based on diagnostic laboratory submissions 12 Oxidised Mannan: A Novel Adjuvant Candidate for Enhancing Immune Responses in Veterinary Rabies Vaccine 18 Insights into artificial waterhole utilisation patterns by elephants and rhinos: Lessons from a South African Nature Reserve 28 Find Rhinos without Finding Rhinos: Active Learning with Multimodal Imagery of South African Rhino Habitats 34 The Contribution of Veterinary Science in Rhino Conservation Association / Vereniging 36 CVC News 37 SAVA News 42 Legal Mews Relax/ Ontspan 39 The warmest place to be Vet's Health / Gesondheid 41 Life Coaching Technical / Tegnies 44 Dental Column Marketplace / Markplein 46 Marketplace Jobs / Poste 47 Jobs / Poste 48 Classifieds / Snuffeladvertensies 12 34 28 Click on the image to access Vetnews CPD articles « nuus•news
Vetnews | September 2025 2 « BACK TO CONTENTS September marks Rabies Month in South Africa, and on the 26th, we join the global community in observing World Rabies Day. Much progress has been made in implementing the National Rabies Strategy, and it is encouraging to note that the South African Veterinary Association, through SAVA-CVC, continues to play a leading role in these efforts. The Battersea Project, primarily in Nelson Mandela Bay, has already enabled approximately 160,000 dog vaccinations since March 2024. The project has also been extended to the Free State and Limpopo provinces. This remarkable achievement is moving South Africa steadily towards the“Zero by 30”target: the elimination of canine-mediated human rabies by the year 2030. Through collaboration with state veterinary services and community veterinary clinics, the project not only provides mass vaccinations but also strengthens community awareness, facilitates outbreak investigations, and supports access to post-exposure prophylaxis. Together, these efforts bring us closer to the goal of achieving at least 70% vaccination coverage and reducing dog populations responsibly. Let us all, whether in state service or private practice, continue to play our part in securing a human-rabies-free future. This past month, I had the privilege of attending Faculty Day at the University of Pretoria, where research presentations reflected both high scientific quality and direct relevance to the profession. The calibre of undergraduate presentations was particularly inspiring, demonstrating not only technical skill but also confidence and clarity in communication. A presentation on the role of veterinary telemedicine, especially in rural practice, resonated deeply as it addressed an urgent and practical challenge for our profession. I also had the pleasure of attending the SASVEPM Congress 2025, where SAVA’s collaboration with SASVEPM, particularly through training linked to the HWSETA programme, was reinforced. I extend my sincere thanks to SASVEPM for their hospitality and professionalism. The recently released report from the Stakeholder Engagement Meeting on Antimicrobial Resistance and Use held in July 2025 highlights several important issues that require SAVA’s continued contribution. The National Department of Agriculture emphasised the need to work towards prohibiting the use of growth promoters, not only to align with international trade requirements but also to safeguard local food safety. Many countries and regions have already banned or restricted the use of antibiotics as growth promoters and may impose import restrictions on animal products originating from countries that do not comply. The meeting further highlighted gaps in surveillance on antimicrobial resistance (AMR) and antimicrobial usage (AMU), noting their impact on compliance with international standards. Concern was raised regarding the continued availability of over-the-counter antimicrobials, which remain a barrier to establishing harmonised veterinary oversight and limit access to high-value global markets. Engagement on this matter will continue, and SAVA remains committed to contributing constructively to national and international discussions. Looking ahead, an important event for the profession is the upcoming SSC Government-Industry Collaboration Workshop (3–4 September 2025), a joint initiative between South Africa and Denmark. This meeting will provide an opportunity to share experiences in strengthening collaboration between the public and private sectors, with a focus on antimicrobial resistance, biosecurity, and controlled disease outbreak management. Further to this, as an association, we must remain mindful of the Animal Health Act and prepare to contribute meaningfully to the forthcoming public consultation process on its regulations. We trust that the National Department of Agriculture will provide adequate opportunity for stakeholder engagement, and in the meantime, I encourage all colleagues to re-familiarise themselves with the provisions of the Act. In October, we look forward to participating in the Agri-SA Atlas at the Future of Agriculture Congress. It promises to be an essential integrative experience, considering the impacts of factors such as climate change on agriculture and a possible way forward. Despite these positive developments, our profession continues to carry the weight of the ongoing absence of a functioning Veterinary Council. This gap has far-reaching implications for governance, oversight, and the regulation of veterinary practice in South Africa. I urge members to remain patient and to allow due process to unfold, as the South African Veterinary Council (SAVC) and the Minister’s office work towards a resolution in the best interests of the profession. I also encourage colleagues to rely only on credible and verified information regarding this matter. SAVA will ensure that all official updates and communications are promptly shared with members. At the same time, SAVA remains steadfast in its commitment to seeking constructive solutions. We have formally expressed our willingness to assist in facilitating a resolution to this critical issue, and we will continue to engage with all relevant stakeholders to help safeguard the integrity and stability of the profession. Finally, I wish to acknowledge V-Tech for their generous sponsorship of my attendance at the World Veterinary Congress in Washington. I extend particular thanks to Dr. Oosthuyse and his team, who continue to provide invaluable support to SAVA, especially in stakeholder communication. It is my sincere hope that this partnership will continue to grow from strength to strength and expand into other areas of collaboration. Colleagues, as we embrace Rabies Month and reflect on our shared achievements, let us also look forward with optimism. May September bring new beginnings—for our profession, for our institutions, and for the communities we serve. v Groetnis! Ziyanda From the President Dear members, To Progress!
Vetnuus | September 2025 3 Vetnuus | August 2024 3 To find out more: Building better practice, together. The co.mpanion partnership is a co.llaborative model that gives you the ownership, support and autonomy you need to build your individual practice’s legacy inside a growing network. co.mpanion is not a corporate body, it is a professional owned and led veterinary model that is right for you if: You are looking for a support structure. You are looking for a better way to exit from or sell your practice. You want to become a shareholder. www.companion.partners Download Value Proposition View Video WhatsApp Sr Dale Parrish
Vetnews | September 2025 4 « BACK TO CONTENTS All this may be very true, but September is also a bit sombre as it is the month we focus on Rabies. When I spoke to Dr Didi Claassen about anything new and exciting in the Rabies world in South Africa, she responded that there is nothing new in Rabies. It has been around for so long that we may have exhausted the topic. Yet there is good news. The province of Mpumalanga has managed to curb the animal-human transmission. They have not reported a single case in the foreseeable past. Definitely a step in the right direction toward the goal of a rabies-free world in 2030. The Global Alliance for Rabies records all events taking place in the world, and it has been reported that South Africa hosts the most events per year. If you are hosting an event, go ahead and log it on their website. https://rabiesalliance.org/world-rabies-day In our district, Mopani, Limpopo, I see that rabies vaccination campaigns have already started, and they are being brought to the people. A great initiative to curb this awful disease. The National Rabies Advisory Group has earmarked the following dates for Rabies updates. Keep an eye out for them and use them in your practice and with your clients: Vetnews also focuses on Rhinos as it is also World Rhino Day, celebrated on September 22, which is for celebrating rhinos and debunking the myth that rhino horn has curative properties. We apologise for the mistake with the birth date of Dr Bruce Fivaz. His birth year is 1946 and not 1940 as indicated. Happy Spring Day!! v Andriette From the Editor Editor’s notes / Redakteurs notas 24-Hour Toll-Free Helpline: 0800 212121 This year on September 28, we are calling on you, me, and our communities to make a difference now! For the first time in its 19-year history, 2025’s theme does not include the word "rabies", showing how well-established this movement has become. Whether you are an individual, part of an organization, or a decision-maker, the time to act is today. • You – Take action in your personal life: vaccinate your dog, educate yourself about how to prevent rabies and Pre- and Post-Exposure Prophylaxis, or advocate for better policies. • Me – Lead by example: inspire others, train professionals, or support rabies elimination efforts in your community. • Community – Work together: organize vaccination campaigns, educate learners and their families, and push for stronger rabies elimination programs. ‘September is the ninth month of the Gregorian calendar, has 30 days, and marks the beginning of autumn in the Northern Hemisphere and spring in the Southern Hemisphere. Its name comes from the Latin word for “seven,” as it was originally the seventh month in the ancient Roman calendar. People born in September are either Virgos or Libras, and the month’s birthstone is sapphire.’
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Vetnews | September 2025 6 « BACK TO CONTENTS Insights into the occurrence of rabies viruses in multi-species animals based on diagnostic laboratory submissions Aurelle Yondo,1 Ben Enyetornye,1 Binu T. Velayudhan1 ABSTRACT Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), primarily affecting the central nervous system of mammals. Understanding the epidemiology of animal rabies is critical for developing effective prevention and control strategies. This study aimed to analyse animal rabies cases received at a veterinary diagnostic laboratory in Georgia, USA, over 5 years (2019–2023), focusing on the most commonly infected species, seasonality trends, and geographical distributions. A total of 1,560 rabies-suspect cases, representing 21 species of animals, were tested using a direct fluorescent antibody test (DFAT). Of 1,560 cases, 5.6% (88/1560) were positive across 11 species, with domestic animals accounting for 17% (15/88) of rabies cases, whereas wildlife species exhibited a higher occurrence of 83% (73/88). 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 included bovine (6.8% [6/88]), feline (5.7% [5/88]), caprine (2.3% [2/88]), and equine (2.3% [2/88]). Positive cases were predominantly detected in submissions from Georgia, with a few additional cases identified in neighbouring states and unknown locations. Furthermore, fall, spring, and summer seasons showed high infection rates compared with winter. Our findings highlight distinct seasonal trends and the significant burden of rabies among wildlife in the Southeastern United States. Editor Hyun Jin Kwun, Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA IMPORTANCE Rabies is a fatal zoonotic viral disease that affects the central nervous system of mammals, including humans. It is transmitted mainly through bites or scratches by infected animals such as dogs, bats, raccoons, and other wild animals. The present study analysed data on clinical specimens submitted to a veterinary diagnostic laboratory for the detection of rabies in domestic and wild animals for a period of 5 years. The study examined a total of 1,560 rabies-suspect cases, representing 21 species of animals tested using the standard direct fluorescent antibody (DFA) assay. Out of 1,560 cases, 5.6% were positive across 11 species, with domestic animals accounting for 17% and wild animals accounting for 83% of the total cases. Different species of wild animals showed a significantly higher incidence of rabies, highlighting the importance of wildlife in spreading rabies to domestic animals and the threat it poses to public health. Rabies is a life-threatening, progressive neurologic viral disease transmitted via the saliva of infected animals, usually through bites or scratches (1–3). It is caused by a bullet-shaped, single-stranded, non-segmented, negative-sense RNA virus belonging to the genus Lyssavirus and the Rhabdoviridae family (4). The rabies virus (RABV) primarily targets the central nervous system of humans and animals, leading to encephalitis with fatal symptoms, including hyperexcitability, autonomic dysfunction, hydrophobia, and aerophobia after an average incubation period of 20–90 days (3, 5, 6). There are rare cases with longer incubation periods, extending up to years, depending on factors such as exposure site, viral load, and host immune response (7, 8). It is a multiple-host pathogen that affects all warm-blooded animals, but dogs and wildlife serve as significant reservoirs for the virus (9, 10). Rabies represents a significant public health threat on every continent except Antarctica (11), with an estimated 60,000 human cases reported annually (12, 13). Although the global burden of rabies seems to have declined over the past three decades, the disease remains a persistent problem for many countries, including developed nations (14). In wildlife, the rabies virus continues to circulate, frequently exposing unvaccinated domestic animals, especially dogs, making control incredibly challenging (15), underscoring the 2030 dog-mediated rabies elimination goals (16). In the USA, approximately 4,000 animal rabies cases are reported annually, with over 90% occurring in wildlife such as skunks, bats, raccoons, and foxes (17). In 2020, 4,090 wildlife and 389 domestic animals tested positive for rabies in the country (18). Human rabies cases in the Americas and Caribbean have been linked to sporadic spillover from wildlife, as widespread preventive measures, such as vaccination, have been implemented for companion animals (19, 20). Each year, more than 4 million Americans report animal bites, with approximately 800,000 seeking medical attention (17). Humans exposed to rabies-positive animals often face long quarantine periods and post-exposure prophylaxis (PEP), causing discomfort and financial burdens to many families (21). Moreover, PEP is expensive and associated with adverse reactions (22). The estimated annual direct and indirect costs of PEP are $1.7 billion and $1.3 billion, respectively (23). This suggests that improving rabies control in wildlife through oral vaccination programs, combined with routine vaccination of companion animals and livestock (24, 25) at a lower cost, could alleviate the burden on animal owners. Given these challenges, constantly updating the epidemiological trends of animal rabies cases submitted to veterinary diagnostic laboratories is crucial to guide the structuring and implementation of preventive and control measures in animals and provide insights
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).
Vetnews | September 2025 8 « BACK TO CONTENTS Leading Article Figure 1 (a) Year-wise distribution of rabies from 2019 to 2023 from cases submitted at Athens Veterinary Diagnostic Laboratory (AVDL) (b) Distribution of positive rabies cases among domestic and wildlife animals over 5 years (2019–2023) from cases submitted at AVDL (c) Wildlife species distribution of rabies over 5 years (2019–2023) from cases submitted at AVDL (d) Domestic species distribution of rabies over 5 years (2019–2023) from cases submitted at AVDL
Vetnuus | September 2025 9 DISCUSSION The study investigated the occurrence of rabies among wild and domestic animals between 2019 and 2023, identified the most affected species, mapped the geographic distribution of positive cases, and analysed rabies trends across the years, considering four different seasons (winter, spring, summer, and fall). In recent years, studies conducted in the United States have reported a significant decrease in the number of rabies-positive cases in 2021, followed by an increase in 2022 (28, 29). Our data reveal a comparable trend over the same period, with the decline observed in 2021 potentially related to decreased rabies surveillance activities during the COVID-19 pandemic (28). Our findings also showed that wildlife species exhibit a significantly higher occurrence of rabies than domestic animals, with 83% of cases (73 out of 88 total cases) reported in wildlife. Likewise, studies conducted in the southeastern United States also observed a higher occurrence of rabies in wildlife compared with domestic animals (30, 31). This higher occurrence in wildlife could be attributed to factors such as larger wildlife population densities, increased interactions between wildlife species, and habitat changes (32, 33). The roles of these factors in the occurrence of rabies were not assessed in this study, which is a limitation. Another factor could be a large sample size, especially for raccoons (n = 253), and the absence of wildlife-targeted vaccination programs in the southeastern United States. In our study, 79 out of 88 positive domestic and wildlife total cases had unknown vaccination history, which suggests a gap in the surveillance of rabies. Recently, challenges in administering oral rabies vaccination (ORV) in skunk populations have been reported (34). Therefore, further investigations could help in implementing more effective control measures, particularly for species like raccoons and skunks, which are known carriers of rabies. In contrast to our findings, domestic animals such as dogs and cats were reported as the most affected by rabies in Brazil, Ukraine, and South Africa (35–37). The high occurrence of rabies in Georgia might be due to the proximity of our laboratory, where we may have received more cases within the state of Georgia than outside the state. This finding can also reflect the presence of RABV in this specific region and help inform targeted rabies control strategies. However, although the geographic distribution in our study was predominantly concentrated in Georgia, the additional cases identified in Florida and Alabama in another study suggest a broader regional spread of the disease across the southeastern United States (30). Leading Article Figure 2: Geographical distribution of positive rabies cases received across the United States Figure 3: Seasonal trend of positive animal rabies cases per year (2019–2023) from cases submitted at AVDL. Seasons were defined as Winter (December–February), Spring (March–May), Summer (June–August) and Fall (September–November) >>>10
Vetnews | September 2025 10 « BACK TO CONTENTS Leading Article Although no statistical significance was observed (P = 0.3387), the rabies infection rates during the fall (6.9%), spring (6%), and summer (5.7%) seasons were higher than the winter (3.8%) seasons, aligning with the highest submission rates. It could be linked to a possible connection between increased activity among wildlife and domestic animals during warmer months and the higher occurrence of rabies (32). During the seasonal peak in the occurrence of rabies that we observed between July and October 2023, the most affected species were raccoons (7.6% [10/131]), whereas tailed deer (2.3% [3/131]), skunks (1.5% [2/131]), bats (0.8% [1/131]), caprine (0.8% [1/131]), and bovine (0.8% [1/131]) were the least affected. It is unknown whether this increase in rabies occurrence was due to a decrease in surveillance activity. In Brazil, equine rabies cases were consistently reported throughout the year, with no clear seasonality, although peaks were noted in certain months due to increased animal interactions (38). It is noteworthy that climate change, with its associated rise in temperature, has been associated with increased rabies cases since animals will be more active and be able to move longer distances in warmer temperatures, thereby potentially spreading the virus to other animals and even humans (32, 39). Moreover, our data confirm that wild animals are more likely to test positive for rabies than domestic animals in the southeastern United States. The most affected species, including raccoons, skunks, white-tailed deer, foxes, bats, and bobcats, further emphasise the significant role of wildlife in the circulation of RABV infection in the region. Although DFAT is the gold standard for rabies testing (40), it has several limitations, including the high rate of inaccurate results due to the requirement of using high-quality brain samples to perform the test. Additionally, the interpretation of the results is very subjective and heavily depends on technicians who need to be highly trained before performing the test while following strict biosafety procedures (41). Access to a necropsy facility is required for proper collection of brain tissues, and a cold chain needs to be maintained to prevent degradation. Such challenges limit DFAT’s use, especially in resource-limited settings. Although the quality of submitted samples was not a constant constraint in our DFAT workflow, it is important to highlight these limitations when analysing rabies data, especially collected in remote areas. Alternative methods, such as real-time polymerase chain reaction (RT-PCR) or LN34 Pan-Lyssavirus RT-PCR assays, could bridge the gap in point-of-care testing as they offer not only a very sensitive and specific diagnostic platform for RABV but also a rapid and costeffective solution (42, 43). Overall, our findings highlight distinct seasonal and geographical trends and the burden of rabies among various animal species. We expect these results would add valuable insights into the literature and public health policymakers in the Southeastern United States and contribute to the battle against rabies. ACKNOWLEDGMENTS We would like to express our appreciation to the UGA-AVDL faculty and staff, especially the virology and serology laboratory team, and Dr. Deborah Keys for the statistical analysis guidance. The authors received no external financial support for the research, authorship, and/or publication of this article. AUTHOR AFFILIATION 1Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA AUTHOR CONTRIBUTIONS Aurelle Yondo, Conceptualisation, formal analysis, methodology, writing - original draft | Ben Enyetornye, Conceptualisation, formal analysis, methodology, writing - original draft | Binu T. Velayudhan, Conceptualisation, Funding acquisition, Investigation, Resources, Supervision, Writing – review and editing ETHICS APPROVAL This study was conducted as a retrospective analysis of specimens submitted to the veterinary diagnostic laboratory and involved no live animals. All data were obtained from existing laboratory records, and any identifiable information was anonymized before analysis to adhere to integrity and confidentiality standards. v References available on request.
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Vetnews | September 2025 12 « BACK TO CONTENTS Oxidised Mannan: A Novel Adjuvant Candidate for Enhancing Immune Responses in Veterinary Rabies Vaccine Rajab Mardani1, Anahita Bahmanje1, Yousef Cheraghi Kazeroni1, Fereydoon Khoshroo1, Bahram Roshanaie2, Tahereh Sadeghche3, Kourosh Pajaie3, Seyed Nezamedin Hosseini4, Delaram Doroud4,*, and Maryam Shahali1,* Departments of 1Viral Vaccines Production, 2Quality Control, and 3Hepatitis B Vaccine Production, Research and Production Complex, Pasteur Institute of Iran, 4Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran Rabies continues to pose a serious public health threat worldwide, with vaccination being the most effective means of prevention. However, commercially available inactivated rabies vaccines often require multiple doses and lack potent adjuvants to enhance their efficacy. This study aimed to investigate the coupling of whole inactivated rabies virus to mannan under oxidising conditions to improve immune responses against a standard rabies vaccine. We explored the conjugation of whole inactivated rabies virus with oxidised mannan (Rab-OxMan) to enhance immune responses. Mice were immunised intraperitoneally with 350 µg of the Rab-OxMan formulation on days 1 and 7. Two weeks after immunisation, serum samples were collected to measure levels of IgG, IgM, and TNF-a using ELISA. The vaccine’s potency was also evaluated using the National Institutes of Health (NIH) assay. Our findings showed a significant increase in IgG levels and a decrease in IgM levels in the Rab-OxMan group compared to the Alumadjuvanted vaccine group (p<0.05). Additionally, TNF-α levels were notably higher in the Rab-OxMan group (p<0.05). Statistical analysis revealed that IgG levels had the highest sensitivity and specificity, with a significant correlation between the measured variables. Importantly, the Rab-OxMan formulation provided 1.8 times greater protection in challenge tests compared to the alum-adjuvanted group. This study is the first to demonstrate that oxidised mannan can serve as a novel adjuvant for veterinary rabies vaccines. The results highlight significant improvements in the immunogenicity and efficacy of rabies vaccines, suggesting a promising strategy for enhancing rabies prevention and potentially reducing the incidence of this deadly disease. INTRODUCTION Rabies is one of humanity’s oldest infectious diseases. The virus claims approximately 60,000 human lives annually and also causes an economic loss of 8.6 billion USD per year globally.1 An estimated number of 10 million people receive post-exposure treatments each year after being exposed to animals suspected to be infected with rabies.2
Vetnuus | September 2025 13 Article The development of the first rabies vaccine by Pasteur successfully reduced the incidence of rabies, but the disease has not been eliminated because it is maintained in many animal reservoirs.3 Many researchers have attempted to produce an affordable and safe rabies vaccine, and the currently recommended inactivated rabies vaccine, adjuvanted with aluminium hydroxide gel, the most common adjuvant, which only induces T helper cell type 2 (Th2) immune responses.4 Therefore, new adjuvants are required to increase the immunogenicity of inactivated rabies vaccines. Various carbohydrates such as β-glucan, mannan, and monophosphoryl lipid A (MPLA) can activate the immune system and induce T helper cell type 1 (Th1) immune responses,5,6; therefore, they are attractive immune adjuvant candidates. They may be used alone or in combination with other adjuvants such as Alum. Carbohydrates can be readily metabolised or degraded in vivo and are less likely to generate long-term toxicity.7 With their biocompatibility, low toxicity and ease of modification, carbohydrates have been studied as carriers for antigen delivery,8 which can often induce immune cell targeting and provide self-adjuvanting activities for a successful vaccination. Mannan, a polysaccharide derived from the structure of plants as well as the cell wall of yeasts, fungi and bacteria, contains mostly a β-1,4-linked mannose backbone with a small number of β-1,6linked glucose and galactose side chain residues.9 The carbohydrate can be recognised through binding with mannose recognition lectins presented on macrophages and other immune cells, which activates the host immune system via a non-self-recognition mechanism.10,11 The recognition initiates a set of signal transduction events leading to cytokine secretion, complement activation and CD8+ T cell activation.12 Although natural carbohydrates can be applied as vaccine components directly,13 in many cases, chemical modification of carbohydrates and/or covalent conjugates of antigens and adjuvants is necessary for enhanced efficacy.14 This can be beneficial in multiple ways, such as prolonged circulation and controlled release, size-induced lymph node targeting, better immune recognition through multivalency, enhanced cell uptake and immune activation. Two strategies (oxidative or reductive) for linking mannan to Antigens have been investigated, which induced drastically different types of immune responses.15,16 The conjugation strategy has been applied in many studies, including vaccines against cancer and influenza, such as breast cancer antigen, Mucin1,17 PCV2 protein of porcine circovirus type 2 virus (PCV2),18 secreted listeriolysin O (LLO) protein of Listeria monocytogenes,19 and inactivated H1N1 influenza virus20 for investigation of the enhancement of the immune responses. Based on previous reports, our studies focused on the investigation of the inactivated rabies virus conjugated to oxidised mannan to its vaccine efficacy. MATERIALS AND METHODS 1. Cell, virus, and mice BHK-21 C13 cells, obtained from the Institute Pasteur (Alborz, Iran), were used in this study. Pasteur strain PV fixed rabies virus, adapted to grow in BHK-21 cells (PV/BHK-21) and provided by the Institut Pasteur (Alborz, Iran), was used throughout this study. All mice were outbred female SW1 sourced from the Research and Production Complex of Pasteur Institute of Iran (Alborz, Iran). All mouse work was conducted at the Animal Laboratory of the quality control department of the Research and Production Complex of Pasteur Institute of Iran (Alborz, Iran), in accordance with an animal ethics application approved by the Iranian Animal Ethics Committee. 2. Cell culture BHK-21 Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) supplemented with fetal Bovine serum (FBS; 5-10%; Invitrogen, USA) and tryptose phosphate (TP; 0.2-0.3% w/v; Invitrogen, USA). 3. Virus production and inactivation BHK-21 cells were infected by the rabies virus strain PV/BHK-21 at a cell concentration of 2-3×106 cells/mL, with a multiplicity of infection (MOI) equal to 0.1/cell in a 10-L bioreactor containing 7 L of DMEM (Invitrogen, USA), supplemented with tryptose phosphate (TP; 0.20.3% w/v; Invitrogen, USA). For the rabies virus production step, pH was maintained at 7.4, pO2 at 30% air saturation, agitation rate at 40 rpm and temperature at 37ºC. The cell suspensions were centrifuged at 750-850 g for 10 min, and the viral supernatants (harvests) were first clarified by filtration through a 0.8-micron filter and then inactivated by 3 mM of Binary ethyleneimine (BEI) (Sigma, USA). 4. Rabies-mannan conjugate Whole inactivated rabies was conjugated to oxidised mannan described in Stambas et al.19 Briefly, mannan (Sigma; USA) (1 mL of 14 mg/mL) in 0.1 M phosphate, pH 6.0, was oxidised with the addition of 0.1 M sodium periodate (100 μL in water) in the dark at 4ºC for 1 hr. The mixture was quenched with 10 µL ethandiol and reacted for a further 30 min as before. The oxidised mannan mixture was passed through a PD10 column (GE Biosciences) pre-equilibrated with 0.05 M bicarbonate, pH 9.0, to remove byproducts. The eluted 2 mL fraction of oxidised mannan (≈7 mg/mL) after void volume (2.5 mL) was collected. Each milliliter of the whole inactivated rabies virus bulk was separately reacted with 0.5 mg/mL Al (OH)3 gel (Croda; Denmark), 100 µg of filter-sterilized mannan (Sigma; USA), or 350 µg of filter-sterilized oxidized mannan for 16 h at 20ºC in 100 rpm and the resulting preparations were analyzed for its vaccine safety and potency. According to previous studies, the periodate oxidation condition for mannan was chosen such that aldehyde residues are generated from only a fraction of oxidised mannose units of the mannan, without affecting its C-type lectin binding activity.21 For a complex antigen such as a whole inactivated rabies virus, we expect the majority of conjugation of mannan aldehyde groups to take place only at the exposed amino groups, forming Schiff base linkages. 5. Inactivity test A 0.03 mL of the inactivated bulk rabies vaccine was intracerebrally administered to each of ten SW1 female mice, with body weights ranging from 11 to 15 g. The animals are observed for 21 days. If more than two animals die during the first 48 hours, the test is repeated. The vaccine complies with the test if, from day 3 to day 21 post-injection, the animals show no signs of rabies, and the immunofluorescence test carried out on the brains of the animals shows no indication of the presence of rabies virus.22 6. Safety test 1 mL of the formulated bulk rabies vaccine was injected intraperitoneally into each of eight female SW1 mice, each weighing 17-22 g. The animals are observed for 21 days. The animals are observed at least daily for 14 days. >>>14
Vetnews | September 2025 14 « BACK TO CONTENTS Article The vaccine complies with the test if no animal shows adverse reactions or dies of causes attributable to the vaccine.22 7. Serological test In the serological test, ten female SW1 mice, each weighing 13-16 g, are used. Each mouse is vaccinated by an intraperitoneal route using 0.5 mL of 1/5 of the recommended dose volume at days 0 and 7. Blood samples are taken 14 days after the first injection, and the sera are tested individually for IgG, IgM, and TNFα by quantitative enzyme-linked immunosorbent assay.23 8. Potency test In addition to Serological assays, potency was analysed according to the National Institutes of Health (NIH) test recommended by the WHO. Mice were immunised on days 0 and 7 with 0.5 mL/dose of all experimental and reference groups via intraperitoneal (IP). Both the experimental vaccines and the international reference standard rabies vaccine were diluted in a serial 5-fold dilution (1/25, 1/125, 1/625). All mice were then challenged on day 14 via an intracerebral administration (IC) of 30 µL rabies strain CVS11 (Pasteur Institute of Iran, Alborz, Iran) containing 42 LD50. Subsequently, mice were observed for another 14 days, and the mortality of mice was recorded to calculate the ED50 that is normalised with the international reference standard vaccine by using the Spearman–Karber formula to obtain a titer in IU NIH/dose. The Ph. Eur. Biological Reference Preparation (BRP) Batch Number 5 for rabies vaccine (inactivated) for veterinary use (EDQM) was used to calibrate the test. The potency is expressed in International Units/mL (IU/mL).24 9. Statistical analysis Statistical analysis was performed using one-way ANOVA using GraphPad Prism (version 8.0, GraphPad Software, CA, USA), and statistical analyses were performed using SPSS Statistics software version 26.0 (IBM, USA). All titrations were carried out in triplicate, and titers are expressed as mean values±standard deviation (SD). The comparison between groups was considered statistically significant if p<0.05 or 0.001. Furthermore, receiver operating characteristic (ROC) curves were utilised to evaluate the sensitivity and specificity of the diagnostic test, while multivariable logistic regression analysis was employed to examine the association between the predictor variables and the outcome variable. RESULTS 1. IgG and IgM assay As shown in Fig. 1A, mice immunized with the conjugate of inactivated rabies virus and oxidized mannan (Rab-OxMan) exhibited a significantly higher IgG titer (12.09±0.94 mg/mL) compared to those receiving mannan (Rab-Man) (11.01±0.65 mg/mL) and the Alumadjuvanted inactivated rabies vaccine (Rab-Al) (7.94±0.71 mg/mL), with a p-value of less than 0.05 indicating statistical significance. The elevated IgG response is promising, as higher IgG levels are often correlated with enhanced protection and long-term immunity against pathogens. Conversely, as illustrated in Fig. 1B, the Rab-OxMan group showed a significantly lower IgM titer (0.54±0.05 mg/mL) than both the Rab-Man (1.03±0.10 mg/mL) and Rab-Al groups (0.72±0.07 mg/mL), with p-values<0.05. This reduction in IgM, while statistically significant, suggests a potential trade-off, as IgM is typically the first antibody produced in response to infection, reflecting early immune activation. These results indicate that although the Rab-OxMan formulation effectively induces a stronger IgG response, which is beneficial for longterm efficacy, it is associated with a reduced IgM response compared to other vaccine formulations. Further investigations are needed to evaluate the longevity of the IgG response and its correlation with long-term protection, as well as to understand the implications of the lower IgM response in the context of overall vaccine efficacy. 2. TNF-α assay In this assay, serum levels of TNFαmeasured by ELISA, were significantly higher in mice vaccinated with Rab-OxMan (47.67±13.1 pg/mL) compared to those immunised with Rab-Man (27.0±8.7 pg/mL) and Rab-Al (24.33±6.2 pg/mL), p<0.05. These results, depicted in Fig. 2, demonstrate that immunisation with the RabOxMan vaccine leads to an increased TNF-α level compared to the other groups, highlighting its potential effectiveness in eliciting a robust immune response. 3. Sensitivity and specificity analysis of the variables The specificity and sensitivity of the sera IgG, IgM and TNF-α levels were assessed using a receiver operating characteristic (ROC) curve. Areas under the curve (AUCs) and p-values were obtained as AUC=0.895 and p<0.001 for IgG, AUC=0.827 and p<0.001 for IgM, and AUC=0.752 and p<0.001 for TNF-α (Fig. 3). According to the results, the sera IgG levels demonstrated the highest sensitivity and specificity compared with other variables. Oxidised Mannan..... <<< 13
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