United Against Rabies Forum: The first 2 years.

Rabies continues to kill an estimated 59,000 people annually, with up to 99% of human cases transmitted by domestic dogs. The elimination of human deaths from dog-mediated rabies is achievable by applying a One Health approach, and the framework to do this is outlined in Zero by 30: the Global Strategic Plan to end human deaths from dog-mediated rabies by 2030. To build on this global goal, and implement the approaches set out in Zero by 30, the United Against Rabies Forum was launched in 2020. This paper gives a review of the objectives, governance, activities and achievements of the United Against Rabies Forum to date. It also outlines ongoing work, and next steps as the United Against Rabies Forum reviews its first 2 years of activities and identifies priority areas for the coming 12 months.

Mitochondrial Dysfunction in Rabies Virus-Infected Human and Canine Brains.

Rabies is a fatal encephalitis caused by the Rabies lyssavirus (RABV). The presence of minimal neuropathological changes observed in rabies indicates that neuronal dysfunction, rather than neuronal death contributes to the fatal outcome. The role of mitochondrial changes has been suggested as a possible mechanism for neuronal dysfunction in rabies. However, these findings are mostly based on studies that have employed experimental models and laboratory-adapted virus. Studies on brain tissues from naturally infected human and animal hosts are lacking. The current study investigated the role of mitochondrial changes in rabies by morphological, biochemical and proteomic analysis of RABV-infected human and canine brains. Morphological analysis showed minimal inflammation with preserved neuronal and disrupted mitochondrial structure in both human and canine brains. Proteomic analysis revealed involvement of mitochondrial processes (oxidative phosphorylation, cristae formation, homeostasis and transport), synaptic proteins and autophagic pathways, with over-expression of subunits of mitochondrial respiratory complexes. Consistent with these findings, human and canine brains displayed elevated activities of complexes I (p < 0.05), IV (p < 0.05) and V (p < 0.05). However, this did not result in elevated ATP production (p < 0.0001), probably due to lowered mitochondrial membrane potential as noted in RABV-infected cells in culture. These could lead to mitochondrial dysfunction and mitophagy as indicated by expression of FKBP8 (p < 0.05) and PINK1 (p < 0.001)/PARKIN (p > 0.05) and ensuing autophagy, as shown by the status of LCIII (p < 0.05), LAMP1 (p < 0.001) and pertinent ultrastructural markers. We propose that altered mitochondrial bioenergetics and cristae architecture probably induce mitophagy, leading to autophagy and consequent neuronal dysfunction in rabies.

Review of Oral Rabies Vaccination of Dogs and Its Application in India.

Oral rabies vaccines (ORVs) have been in use to successfully control rabies in wildlife since 1978 across Europe and the USA. This review focuses on the potential and need for the use of ORVs in free-roaming dogs to control dog-transmitted rabies in India. Iterative work to improve ORVs over the past four decades has resulted in vaccines that have high safety profiles whilst generating a consistent protective immune response to the rabies virus. The available evidence for safety and efficacy of modern ORVs in dogs and the broad and outspoken support from prominent global public health institutions for their use provides confidence to national authorities considering their use in rabies-endemic regions. India is estimated to have the largest rabies burden of any country and, whilst considerable progress has been made to increase access to human rabies prophylaxis, examples of high-output mass dog vaccination campaigns to eliminate the virus at the source remain limited. Efficiently accessing a large proportion of the dog population through parenteral methods is a considerable challenge due to the large, evasive stray dog population in many settings. Existing parenteral approaches require large skilled dog-catching teams to reach these dogs, which present financial, operational and logistical limitations to achieve 70% dog vaccination coverage in urban settings in a short duration. ORV presents the potential to accelerate the development of approaches to eliminate rabies across large areas of the South Asia region. Here we review the use of ORVs in wildlife and dogs, with specific consideration of the India setting. We also present the results of a risk analysis for a hypothetical campaign using ORV for the vaccination of dogs in an Indian state.

Investigation of protective level of rabies antibodies in vaccinated dogs in Chennai, India.

BACKGROUND: Rabies is still endemic in India causing an estimated 20,000 human deaths a year. Free roaming dogs and unvaccinated owned dogs play a major role in the maintenance of the disease. Dog vaccination is the most crucial aspect of rabies prevention and control strategies; therefore vaccine immunogenicity and longevity are important determinants of the efficiency of rabies control efforts. METHODS: In this study at Madras Veterinary College, India, a total of 297 serum samples were collected from owned dogs that were vaccinated against rabies. Data regarding age, gender, breed, neuter status and last date of vaccination were collected at the time of blood collection. The level of rabies virus neutralising antibodies in the sera of these dogs was measured through rapid focus fluorescence inhibition test. The factors associated with protective level of rabies antibodies in vaccinated dogs were investigated through multivariable regression analysis. RESULTS: This cross-sectional investigation shows that only 40% (119/297) of the all the dogs in the study showed presence of protective level of anti-rabies antibodies, and 40% (72/180) of the dogs vaccinated within the last year showed presence of protective levels of antibodies causing concern about rabies vaccine quality and its impact on rabies control. The study also shows that older and neutered dogs are more likely to have protective titre among vaccinated dogs, while non-descript breed dogs are less likely to have a protective titre compared to pure breeds. CONCLUSION: In this study 60% (108/180) of young prima dogs and adult dogs did not show protective levels of antibodies within the year of last rabies vaccination, although they had previous vaccination history. This high percentage of apparent non-responders is a cause of concern of administration, distribution, storage, potency and quality management of vaccines in India.

Portable Rabies Virus Sequencing in Canine Rabies Endemic Countries Using the Oxford Nanopore MinION.

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION's potential for low-cost, high volume sequencing of pathogens in locations with limited resources.

Towards rabies elimination in the Asia-Pacific region: From theory to practice.

Rabies is a major neglected zoonotic disease and causes a substantial burden in the Asian region. Currently, Pacific Oceania is free of rabies but enzootic areas throughout southeast Asia represent a major risk of disease introduction to this region. On September 25-26, 2019, researchers, government officials and related stakeholders met at an IABS conference in Bangkok, Thailand to engage on the topic of human rabies mediated by dogs. The objective of the meeting was focused upon snowballing efforts towards achieving substantial progress in rabies prevention, control and elimination within Asia by 2030, and thereby to safeguard the Pacific region. Individual sessions focused upon domestic animal, wildlife and human vaccination; the production and evaluation of quality, safety and efficacy of existing rabies biologics; and the future development of new products. Participants reviewed the progress to date in eliminating canine rabies by mass vaccination, described supportive methods to parenteral administration by oral vaccine application, considered updated global and local approaches at human prophylaxis and discussed the considerable challenges ahead. Such opportunities provide continuous engagement on disease management among professionals at a trans-disciplinary level and promote new applied research collaborations in a modern One Health context.

Development of a Non-Meat-Based, Mass Producible and Effective Bait for Oral Vaccination of Dogs against Rabies in Goa State, India.

Introduction: To achieve the global goal of canine-mediated human rabies elimination by 2030 there is an urgent need to scale-up mass dog vaccination activities in regions with large dog populations that are difficult to access; a common situation in much of India. Oral rabies vaccination may enable the vaccination of free-roaming dogs that are inaccessible to parenteral vaccination, and is considered a promising complementary measure to parenteral mass dog vaccination campaigns. WHO and OIE have published detailed minimum requirements for rabies vaccines and baits to be used for this purpose, requiring that baits must not only be well-accepted by the target population but must also efficiently release the vaccine in the oral cavity. For oral rabies vaccination approaches to be successful, it is necessary to develop baits which have a high uptake by the target population, are culturally accepted and amenable to mass production. The aim of this study was to compare the interest and uptake rates of meat-based and an egg-based prototype bait constructs by free roaming dogs in Goa, India. Methods: Three teams randomly distributed two prototype baits; an egg-flavoured bait and a commercial meat dog food (gravy) flavoured bait. The outcomes of consumption were recorded and compared between baits and dog variables. Results: A total of 209 egg-bait and 195 gravy-bait distributions were recorded and analysed. No difference (p = 0.99) was found in the percentage of dogs interested in the baits when offered. However, significantly more dogs consumed the egg-bait than the gravy-bait; 77.5% versus 68.7% (p = 0.04). The release of the blue-dyed water inside the sachet in the oral cavity of the animals was significant higher in the dogs consuming an egg-bait compared to the gravy-bait (73.4% versus 56.7%, p = 0.001). Conclusions: The egg-based bait had a high uptake amongst free roaming dogs and also enabled efficient release of the vaccine in the oral cavity, whilst also avoiding culturally relevant materials of bovine or porcine meat products.

Evaluation of an Immunochromatographic Assay as a Canine Rabies Surveillance Tool in Goa, India.

Rabies is a fatal zoonotic disease transmitted by the bite of a rabid animal. More than 95% of the human rabies cases in India are attributed to exposure to rabid dogs. This study evaluated the utility of a lateral flow immunochromatographic assay (LFA) (Anigen Rapid Rabies Ag Test Kit, Bionote, Hwaseong-si, Korea) for rapid post mortem diagnosis of rabies in dogs. Brain tissue was collected from 202 animals that were screened through the Government of Goa rabies surveillance system. The brain tissue samples were obtained from 188 dogs, nine cats, three bovines, one jackal and one monkey. In addition, 10 dogs that died due to trauma from road accidents were included as negative controls for the study. The diagnostic performance of LFA was evaluated using results from direct fluorescence antibody test (dFT); the current gold standard post mortem test for rabies infection. Three samples were removed from the analysis as they were autolysed and not fit for testing by dFT. Of the 209 samples tested, 117 tested positive by LFA and 92 tested negative, while 121 tested positive by dFT and 88 tested negative. Estimates of LFA sensitivity and specificity were 0.96 (95% CI 0.91-0.99) and 0.99 (95% CI 0.94-1.00), respectively. The LFA is a simple and low-cost assay that aids in the rapid diagnosis of rabies in the field without the need for expensive laboratory equipment or technical expertise. This study found that Bionote LFA has potential as a screening tool in rabies endemic countries.

Priorities in applied research to ensure programmatic success in the global elimination of canine rabies.

The elimination of human rabies mediated by dogs is attainable in concept, based upon current sensitive and specific diagnostic methods, existing safe and effective human and veterinary vaccines and a sound virological, pathological and epidemiological understanding of the disease. Globally, all developed countries achieved this goal. Regionally, major progress occurred throughout the Americas. However, less advancement is evident in Africa and Asia. Our objective was to concentrate upon those salient improvements to extant tools and methods over the next five years which could assist and simplify the task for both those developing countries that have already begun the process, as well as other localities in the earlier stages of consideration. We considered several categories of applied research which could be accomplished in the short term, based upon the available scientific evidence and recent recommendations from subject matter experts and key opinion leaders, focused upon perceived major limitations to prior program success. Areas of concentration included: laboratory-based surveillance, pathogen detection and characterization; human rabies prophylaxis; veterinary biologics; implementation of canine vaccination; and oral vaccination of free-ranging community dogs. Further real-time application in these core areas with proven techniques and technology would simplify attaining not only the global goal focused subtly upon human mortality, but the actual elimination of canine rabies as well.

Silver nanoparticle-specific mitotoxicity in Daphnia magna.

Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag(+) and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO3 and Ag NPs (130-650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag(+). This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag(+) was more efficient than Ag NPs at displacing Na(+) within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.

Toxicity of soybean rust fungicides to freshwater algae and Daphnia magna.

Soybeans are intensively grown over large swaths of land in the Midwestern US. Introduction of the pathogenic fungus responsible for Soybean Rust (Phakopsora pachyrhizi) will likely result in a significant increase in the environmental load of strobilurin and conazole fungicides. We determined the toxicity of six such fungicides to the unicellular algae Pseudokirchneriella subcapitata and the aquatic invertebrate, Daphnia magna. We found that levels of concern of some fungicides were lower than annual average runoff concentrations predicted for Indiana. Our results suggest that pyraclostrobin and propiconazole, and to a lesser extent tebuconazole, may cause impacts to algae and daphnids in areas where soybeans are intensively grown. More studies are needed to describe the ecological effects of sublethal exposures to these fungicides, as well as monitoring environmental concentrations in watersheds where these fungicides are applied to soybeans.