Collection OverviewRabies is an acute progressive encephalitis caused by a lyssavirus. The case fatality of this zoonosis is the highest for any infectious disease. Although laboratory techniques on rabies have progressed greatly over the last century, typically such information falls in disparate locations and is often unavailable for easy access by those with the greatest need. Hence, the opportunity for a visual, methodological catalog is critical for basic research in pathobiology, immunology and molecular bio… Show Morelogy, as well for applied diagnostics, prophylaxis, therapy and intervention. The time is opportune because a global program is underway for the elimination of human rabies transmitted by dogs through mass canine vaccination, requiring multiple tests for risk mitigation and measurement of programmatic success. Advances have occurred in pathogen discovery, characterization, novel biologics and anti-virals. Based on in vitro and animal models, hope has arisen not only for immunization after viral exposure but for potential treatment after illness. Besides direct application to humans and domestic animals, efficacious vaccines may also be distributed to free-ranging wildlife en masse via vaccine-laden baits. The objective of this collection is to: share standardized protocols for detection, characterization and response; promote broader global access to existing methods; produce open networks for research collaborations anew; and ultimately create a pathway for an evidence-based approach for additional knowledge on this neglected disease. We trust this approach will encourage a myriad of scientists to participate in this global endeavor in a trans-disciplinary manner, towards renewed biomedical progress in human and veterinary science and conservation biology. Show Less
Novel procedure of rabies test for animals by detecting viral antigens stably in Merkel cells at the follicle-sinus complexes of muzzle skins
Rabies is a zoonotic disease caused by lyssaviruses which are transmitted to humans via rabid animals and causes an acute encephalomyelitis after a variable incubation period, and also is the highest case fatality rate of any currently recognized infectious disease. Effective surveillance and monitoring of rabies susceptible animals is a key activity to the assessment of exposed persons. However, the dissection of the head for rabies testing in the laboratory is still laborious with a high… Show More risk of virus exposure, and requires expensive equipment. Skin biopsies were reported useful for ante- and postmortem diagnosis of humans. In animals, the tactile hair, known as the follicle-sinus complex (FSC), has enough sensory nerve endings and viral antigens were clearly localized in Merkel cells (MCs) of rabid animals. This study describes an alternative postmortem rabies test for routine laboratory practices with low-cost, simple and less virus-exposure risks. Formalin-fixed viral antigens in the skin of rabid animals were detected immunohistochemically and pathologically examined in MCs at the FSCs embedded in paraffin blocks. Show Less
Use of mobile technology in the enhanced implementation of mass dog vaccination interventions
Many high income nations have successfully eliminated rabies through annual mass dog vaccination campaigns implemented comprehensively across the country. This requires expertise in project planning, cartography and logistics, the coordination of hundreds of vaccination teams and capacity to aggregate, review and analyse large data sets. There are few low and middle income countries that have demonstrated that this can be achieved at the national scale, let alone refined and sustained to achieve… Show More rabies elimination.
The past decade has seen rapid growth in the use of mobile technology to overcome challenges in conducting public health initiatives in resource limited settings. A smartphone App and website interface have been developed to support the implementation, monitoring and evaluation of mass dog vaccination campaigns.
The project area is first divided into working zones, displayed in the website as polygons on Google Maps. Each working zone contains approximately enough dogs for teams to complete vaccinations in a few days. The project manager then systematically assigns polygons to vaccination teams working in the field. Each team’s assigned region(s) are loaded to the phone and displayed on Google Maps within the App, with a current location marker helping them to navigate inside their assigned area. Every dog vaccinated is recorded in the app, automatically capturing time, date, user and GPS data. Additional information about ownership, confinement, age, sex and health of the animal may also be recorded. Each day the vaccination data are synchronized to the cloud server where it can be accessed through the website for review by the project manager on summary maps. Teams are then further directed using polygon assignment based on their geographic progress.
The system has been used to record over 1 million dog vaccinations in three continents, including national scale in Haiti and India State scale in Goa. Show Less
IN-VITRO TESTS FOR RABIES VACCINE POTENCY TESTING
he growing global concern for animal welfare is encouraging manufacturers and National Control Laboratories (OMCLs) in the “3Rs strategy” for the “Replacement, Reduction and Refinement” of laboratory animal testing. Development of in vitro approaches are recommended at the WHO and European levels as alternatives to the NIH test for evaluating rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute t… Show Morehe major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test using a Neutralizing Monoclonal Antibody (mAb-D1) recognizing the trimeric form of the glycoprotein has been developed to determine the content of the native folded trimeric glycoprotein along production of vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future. The method is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein. The mAb-D1 is used for both coating and detection, which allows to select only the trimers of glycoprotein, i.e. the immunogenic RABV antigen, present in the vaccine batch. The potentially denaturated monomers of glycoprotein (with only one binding site available) cannot be captured and detected by the same mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again labelled with peroxidase, which is revealed in the presence of substrate and chromogen. Comparison of absorbance measured for the tested vaccine and the reference vaccine allows the determination of the glycoprotein content. Show Less
The Rapid Fluorescent Focus Inhibition Test and Adaptions
Abstract: The Rapid Fluorescent Focus Inhibition Test (RFFIT) has been in use since the first publication of the assay in 1973, quickly becoming the gold standard for measurement of rabies virus neutralizing antibodies (RVNA). Because the test system mimics the virus-antibody-cell interactions that determine the function of the RVNA (prevention of infection), it is the best ass… Show Moreay to determine the level of response. Since 1973, the assay has been modified for specific purposes, such as small sample size or need for higher sensitivity and specificity. The RFFIT is a cell-based assay requiring live RABV and a permissive cell line; a fluorescence microscope; and performance in Biosafety Level 2 or 3 facilities. Selection and maintenance of critical reagents as well as meticulous performance of the assay steps are essential for accurate and precise results. New rabies biologics, such as monoclonal antibodies to replace human rabies immune globulin and mRNA vaccines require demonstration of broad specificity of the protective immunity by licensing agencies. To accomplish this, the RFFIT must be adapted and these adaptions must be qualified to GXP/ICH level quality standards. In addition, large number of samples are required for clinical trial studies. These requirements (assay adaptions, high quality standards, and high throughput capability) are challenging for a traditionally manual assay with subjective read-out. This paper presents the performance of the original RFFIT as well as adaptions for the following purposes: increased precision and broad specificity; the validation of each; and comparison of quality standards employed for different purposes. Show Less
Iophenoxic Acid as a biological marker for oral rabies vaccination in the small Indian mongoose
The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in parts of the Caribbean, such as Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and… Show More various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. However, this strategy may difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (ethyl -IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (methyl-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to 8 weeks post offering. Sera were analyzed for ethyl- or methyl-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for ethyl- and methyl-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses, but due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations. Show Less
Quantification of rabies virus in different brain structures of livestock
Bovine Paralytic Rabies (BPR) is of great economic importance throughout Latin America. This viral encephalitis produces significant losses to the livestock industry and poses a major zoonotic risk. For rapid and sensitive diagnosis, selection of the best CNS sample is critical. The objective of our work was to utilize a laboratory protocol to quantify the relative number of rabies virus (RV) particles detected in different anatomical portions of the bovin… Show Moree brain using quantitative RT-PCR (RT-qPCR). RT-qPCR can detect a specific number of gene copies, as the particular fluorescence emitted after amplification is directly proportional to the nucleic acid in the sample. Some advantages include a short time for performance (2 hours), less risk of contamination, the possibility of identifying several pathogens simultaneously and besides use of CNS tissue, the opportunity to detect viral genome in saliva and other fluids much easier than using other techniques. For this study, 34 brains of rabid cattle were divided into six anatomical structures: the Ammon’s horn, cerebellum, cortex, medulla, pons and thalamus. All brains were positive for RV antigens by the direct immunofluorescence test. The same anatomical structures were obtained from 12 negative bovine brains. RNA was extracted from each structure and used for RT-qPCR. The assay was implemented to determine the number of RV copies using an in vitro transcript gene nucleoprotein. The standard curve used to quantify viral RNA showed 100% efficiency and 0.99 of linearity. Results showed that the portion of the CNS that is best to perform to select for the detection of RV includes cortex, medulla and thalamus, as they these structures had the highest amount of RV detected. The test showed 100% specificity: all positives were detected and no false positives with controls. This method can be used to detect the presence of RV in samples for BPR diagnosis with a minimum amount of RV, up to 30 copies. Show Less
Pan-lyssavirus SYBR real time RT-PCR for rabies diagnosis
Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilised for decades to detect rabies, but have only recently been accepted by the OIE as a method to confirm rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimising the risk of contamination during set up. SYBR real-time RT-PCR assays do not require… Show More expensive probes, minimising the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera, rather than specific to just one virus species, are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay which detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with melt curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive detection of animal and human rabies cases. Show Less
Evaluation of a commercial blocking ELISA for the detection of rabies virus antibodies in North American wildlife sera
Rabies lyssavirus (RV) causes an important global zoonosis with an estimated 59,000 human deaths annually. The majority of human cases are transmitted by domestic dogs. Human fatalities within North America are infrequent, yet RV circulates naturally and independently in multiple bat and carnivore reservoirs, resulting in human exposures and prophylaxis. Testing for RV antibodies (RVA) is a key diagnostic activity, widely recognized as a measure of resistance to lethal i… Show Morenfection. Demonstration of RVA is important for studies addressing individual level immune responses in experimental pathogenesis and vaccination studies, as well as population level immunity due to natural RV exposures or vaccination activities targeting reservoir hosts for disease control and prevention. While neutralization assays have historically been considered the gold standard for measurement of RVA, ELISA-based assays are gaining in popularity and may allow improved standardization across laboratories. We evaluated the sensitivity and specificity of a commercial blocking ELISA (BioPro® ELISA kit, O.K. Servis, Prague, Czech Republic) for detection of RVA in comparison to a neutralization assay, using 1,131 sera from studies involving five wild carnivore reservoirs in North America. Specificity of RVA detection was greater than 90% across all studies in comparison to neutralization data. Sensitivity was greater than 90% in a captive vaccination study with striped skunks (Mephitis mephitis) and active surveillance for natural RV exposure in small Indian mongoose (Herpestes auropunctatus), but lower than 45% in a captive vaccination study with raccoons (Procyon lotor), and monitoring of oral rabies vaccination activities targeting raccoons and coyotes (Canis latrans). Overall, the commercial ELISA kit exhibited greater utility in the demonstration of RVA in captive experimental studies and natural RV exposures. Additional kit optimization is necessary for use with certain wild carnivore reservoirs and for monitoring oral rabies vaccination programs in North America. Show Less
Development and validation of the pan-lyssavirus nested RT-PCR for diagnosis of rabies
The pan-lyssavirus nested RT-PCR (RT-nPCR) has been developed to amplify a part of the N gene of lyssaviruses and verified as a highly specific diagnostic tool for detection of all 16 confirmed lyssavirus species classified by ICTV and 2 tentative novel lyssavirus species (Kotalahti bat lyssavirus and Taiwan bat lyssavirus). The validation using 9624 brain specimens collected from different animal species with multiple geographical… Show More origins in China showed that the RT-nPCR had 98.18% sensitivity and 100% specificity in comparison with the “gold standard” direct florescent antibody test (FAT). Furthermore, the RT-nPCR is more suitable than the FAT and mouse inoculation test (MIT) for detection in highly decayed brain tissues. This method provides a convenient alternative for diagnosis of rabies and has been approved by the National Technical Standardization Committee of Animal Health of China as a National Standard (GB/T20111796-t-326). Show Less
Lateral flow test for simple animal rabies diagnosis in resource-limited settings.
High functioning continuous rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for successful sustainable disease control. To date, animals suspected as rabid must have a post-mortem confirmation using either classical or molecular laboratory methods. This necessitates prompt submission of diagnostic samples to a functioning and accessible laboratory. However, in most endemic areas, animal rabies diagnosi… Show Mores is restricted to central veterinary laboratories (CVLs), and this poor availability of surveillance infrastructure leads to serious under reporting of disease for rural and remote areas. Therefore, existing surveillance data primarily reflects the rabies situation in urban areas. In low- and middle-income countries, point-of-care rabies diagnosis is critical to improve disease surveillance. Several new low-technical diagnostic protocols have been recently developed offering opportunities to establish and extend rabies diagnosis in remote areas through decentralized laboratories. We present here an immunodiagnostic test (RIDT, Anigen/Bionote Inc.) which can easily detect rabies virus and other lyssaviruses. The principle of the test is simple: dissolved brain material is applied on a test strip where gold-conjugated antibodies bind specifically to rabies virus antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be later extracted. This allows the test strip, rather than the original infectious sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. The advantages of the lateral flow device are numerous: the test is rapid, easy-to-use, and low-cost but does not require any laboratory infrastructure, such as microscopy or cold-chain compliance. Previous studies have demonstrated the high potential of this test in the African context where rabies is listed as a priority zoonotic disease. Show Less
High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue
Visualization of infection events in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ samples provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricte to thin sections obtained from paraffin-embedded or frozen samples. Because of the limited 2D image plane provided… Show More by thin sections, crucial information on the complex structure of a respective organ as well as on the compartment and the surrounding cellular context of the infection environment is lost. However, modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the organ tissue to organic solvents, it becomes optically transparent, which reduces light scattering, while simultaneously matching the sample’s refractive indices. Thus, in combination with high working distance objectives, large tissue sections up to 1 mm in size can be imaged and visualized by conventional confocal laser scanning microscopy. Here, we describe a protocol to apply tissue clearing-mediated deep tissue imaging to visualize rabies virus distribution in infected brains to study topics such as virus pathogenesis, spread, tropism, and neuroinvasion. Show Less
Laboratory standard operating procedure of lyssavirus surveillance on bat (Chiroptera) population in Taiwan
Viruses within the genus Lyssavirus are zoonotic pathogens and at least seven lyssavirus species are are associated with human cases. Because bats are the natural reservoirs of most lyssaviruses, to understand the ecology of these viruses in bats in Taiwan, a lyssavirus surveillance program of bats has been conducted since 2008. For occupational safety concerns, all laboratory workers handle bat specimens only after receiving pre-exposure rabies prophylaxis (PrEP). All procedures should be perfo… Show Morermed in biosafety level 2 (or above) laboratories and workers should wear proper personal protective equipment (PPE). We cooperate with bat conservation non-governmental organizations and local animal disease control centers to collect bats which showed weakness, signs of illness or were found dead. It is not recommended to euthanize apparently healthy bats for surveillance purposes. Bat species were identified morphologically. Brain tissues of the collected bats were obtained through necropsy and subjected to the direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detecting lyssavirus antigens. When performing the FAT, using at least two brands of commercially available rabies diagnosis conjugates were recommended. With regard to the RT-PCR, two sets of pan-lyssavirus primers (JW12/N165-146, N113F/N304R) based on the lyssavirus nucleoprotein (N) gene and phosphoprotein (P) gene, respectively, were used to amplify the partial sequence of lyssavirus. People who submitted the samples usually received results within a week. We hope the rapid feedback encourages the public to keep sending samples to the laboratory. This surveillance allowed us to assess lyssaviruses and other zoonotic agents in wildlife. Such findings may educate the public, health professionals and scientists to acknowledge the potential risk when they contact bats and other wildlife. Show Less
Rabies necropsy techniques on small and large specimens
Safe and efficient necropsy techniques are essential qualities for all rabiesdiagnostic laboratories. Determining the necessary personal protective equipment (PPE) requires evaluation of the necropsy space, instruments in use, and type of specimens that may be submitted. The New York State Department of Health receives between 6,000 and 8,000 specimens annually ranging from juvenile bats to intact submissions from draft horses and adult cattle. A… Show Morell specimens are necropsied in fume hoods, or in the case of Eastern Equine Encephalitis Virus (EEEV) suspect specimens, a specially designed class II biosafety cabinet. The purpose of this video is to demonstrate the PPE required in our laboratory, two different necropsy techniques used on small animals, and the removal of the cerebellum and brainstem from large animals using the opening at the foramen magnum. Show Less
A Protocol for Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test
Laboratory-based surveillance is integral for rabies prevention, control, and management efforts. While the direct fluorescent antibody test (DFA) is the gold standard for rabies diagnosis, there is an ongoing need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for a direct rapidimmunohistochemical test … Show More(DRIT) as an alternative, field-based testing option that utilizes simple light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses monoclonal or polyclonal antibodies conjugated to biotin, a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately one hour, a sample can be collected, tested, and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa and Europe have illustrated sensitivity and specificity by the DRIT, approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture’s Wildlife Services Program has conducted large-scale enhanced rabies surveillance efforts, using the DRIT to test >92,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabiessurveillance, prevention and control programs globally. Show Less