The role of a mechanistic host in maintaining arctic rabies variant distributions: Assessment of functional genetic diversity in Alaskan red fox (Vulpes vulpes).

Populations are exposed to different types and strains of pathogens across heterogeneous landscapes, where local interactions between host and pathogen may present reciprocal selective forces leading to correlated patterns of spatial genetic structure. Understanding these coevolutionary patterns provides insight into mechanisms of disease spread and maintenance. Arctic rabies (AR) is a lethal disease with viral variants that occupy distinct geographic distributions across North America and Europe. Red fox (Vulpes vulpes) are a highly susceptible AR host, whose range overlaps both geographically distinct AR strains and regions where AR is absent. It is unclear if genetic structure exists among red fox populations relative to the presence/absence of AR or the spatial distribution of AR variants. Acquiring these data may enhance our understanding of the role of red fox in AR maintenance/spread and inform disease control strategies. Using a genotyping-by-sequencing assay targeting 116 genomic regions of immunogenetic relevance, we screened for sequence variation among red fox populations from Alaska and an outgroup from Ontario, including areas with different AR variants, and regions where the disease was absent. Presumed neutral SNP data from the assay found negligible levels of neutral genetic structure among Alaskan populations. The immunogenetically-associated data identified 30 outlier SNPs supporting weak to moderate genetic structure between regions with and without AR in Alaska. The outliers included SNPs with the potential to cause missense mutations within several toll-like receptor genes that have been associated with AR outcome. In contrast, there was a lack of genetic structure between regions with different AR variants. Combined, we interpret these data to suggest red fox populations respond differently to the presence of AR, but not AR variants. This research increases our understanding of AR dynamics in the Arctic, where host/disease patterns are undergoing flux in a rapidly changing Arctic landscape, including the continued northward expansion of red fox into regions previously predominated by the arctic fox (Vulpes lagopus).

A recombinase polymerase amplification assay for rapid detection of rabies virus.

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

Rabies control in Liberia: Joint efforts towards zero by 30.

Despite declaration as a national priority disease, dog rabies remains endemic in Liberia, with surveillance systems and disease control activities still developing. The objective of these initial efforts was to establish animal rabies diagnostics, foster collaboration between all rabies control stakeholders, and develop a short-term action plan with estimated costs for rabies control and elimination in Liberia. Four rabies diagnostic tests, the direct fluorescent antibody (DFA) test, the direct immunohistochemical test (dRIT), the reverse transcriptase polymerase chain reaction (RT-PCR) assay and the rapid immunochromatographic diagnostic test (RIDT), were implemented at the Central Veterinary Laboratory (CVL) in Monrovia between July 2017 and February 2018. Seven samples (n=7) out of eight suspected animals were confirmed positive for rabies lyssavirus, and molecular analyses revealed that all isolates belonged to the Africa 2 lineage, subgroup H. During a comprehensive in-country One Health rabies stakeholder meeting in 2018, a practical workplan, a short-term action plan and an accurately costed mass dog vaccination strategy were developed. Liberia is currently at stage 1.5/5 of the Stepwise Approach towards Rabies Elimination (SARE) tool, which corresponds with countries that are scaling up local-level interventions (e.g. dog vaccination campaigns) to the national level. Overall an estimated 5.3 - 8 million USD invested over 13 years is needed to eliminate rabies in Liberia by 2030. Liberia still has a long road to become free from dog-rabies. However, the dialogue between all relevant stakeholders took place, and disease surveillance considerably improved through implementing rabies diagnosis at the CVL. The joint efforts of diverse national and international stakeholders laid important foundations to achieve the goal of zero dog-mediated human rabies deaths by 2030.

Epidemiology and preclinical management of dog bites among humans in Wakiso and Kampala districts, Uganda: Implications for prevention of dog bites and rabies.

In rabies endemic areas, appropriate management of dog bites is critical in human rabies prevention. Victims must immediately wash bite wound for 15 minutes with water, soap, and a disinfectant before seeking medical care. This study investigated the epidemiology of dog bites and the determinants of compliance to these pre-clinical guidelines requirements among dog bite victims from high rabies-burden areas of Wakiso and Kampala, Uganda. An explanatory sequential mixed-methods study design was used. Quantitative data were collected from 376 dog-bite patients at two healthcare facilities. Qualitative data were also collected through 13 in-depth interviews with patients, healthcare workers, herbalists, and veterinarians. Qualitative data were analyzed using a deductive thematic approach. Generalized linear models were used to determine factors associated with compliance. Nearly half (190, 51%) of the patients were from Wakiso District and 293 (77.9%) had grade II wounds. Most of the wounds (171, 45.5%) were on the legs. Two-thirds of the bites occurred in public places. Only 70 (19%) of the bite patients had complied with pre-clinical guidelines. Nearly half of the patients had applied substances that were not recommended e.g. herbs (47/193), antiseptics (46/193), "black stone" (25/193), and unknown creams (10/193). Factors negatively associated with compliance included: being aged 15 years or older, adjPR = 0.70 (0.47-0.92) and knowing the dog owner, adjPR = 0.65 (0.36-0.93). However, attainment of secondary or higher education, adjPR = 1.76 (1.24-3.79), being in employment, adjPR = 1.48 (1.09-2.31), perception that the dog was sick, adjPR = 1.47 (1.02-2.72) and knowledge about the dog's subsequent victim(s) adjPR = 0.35 (0.17-0.70) were positively associated with compliance. High occurrence of dog bites in public places by free-roaming dogs suggests the need for deliberate promotion of responsible dog ownership. Additionally, targeted health education may be required to improve the low compliance to pre-clinical guidelines.

Economic and feasibility comparison of the dRIT and DFA for decentralized rabies diagnosis in resource-limited settings: The use of Nigerian dog meat markets as a case study.

BACKGROUND: Rabies lyssavirus (RABV) is the aetiologic agent of rabies, a disease that is severely underreported in Nigeria as well as elsewhere in Africa and Asia. Despite the role that rabies diagnosis plays towards elucidating the true burden of the disease, Nigeria-a country of 180 million inhabitants-has a limited number of diagnostic facilities. In this study, we sought to investigate two of the World Organization for Animal Health (OIE)-recommended diagnostic assays for rabies-viz; the direct fluorescent antibody test (DFA) and the direct rapid immunohistochemical test (dRIT) in terms of their relative suitability in resource-limited settings. Our primary considerations were (1) the financial feasibility for implementation and (2) the diagnostic efficacy. As a case study, we used suspect rabies samples from dog meat markets in Nigeria. METHODS/PRINCIPAL FINDINGS: By developing a simple simulation framework, we suggested that the assay with the lowest cost to implement and routinely use was the dRIT assay. The costs associated with the dRIT were lower in all simulated scenarios, irrespective of the number of samples tested per year. In addition to the cost analysis, the diagnostic efficacies of the two assays were evaluated. To do this, a cohort of DFA-positive and -negative samples collected from dog meat markets in Nigeria were initially diagnosed using the DFA in Nigeria and subsequently sent to South Africa for diagnostic confirmation. In South Africa, all the specimens were re-tested with the DFA, the dRIT and a quantitative real-time polymerase chain reaction (qRT-PCR). In our investigation, discrepancies were observed between the three diagnostic assays; with the incongruent results being resolved by means of confirmatory testing using the heminested reverse transcription polymerase reaction and sequencing to confirm that they were not contamination. CONCLUSIONS/SIGNIFICANCE: The data obtained from this study suggested that the dRIT was not only an effective diagnostic assay that could be used to routinely diagnose rabies, but that the assay was also the most cost-effective option among all of the OIE recommended methods. In addition, the results of our investigation confirmed that some of the dogs slaughtered in dog markets were rabies-positive and that the markets posed a potential public health threat. Lastly, our data showed that the DFA, although regarded as the gold standard test for rabies, has some limitations-particularly at low antigen levels. Based on the results reported here and the current challenges faced in Nigeria, we believe that the dRIT assay would be the most suitable laboratory test for decentralized or confirmatory rabies diagnosis in Nigeria, given its relative speed, accuracy, cost and ease of use.

Human Rabies - Virginia, 2017.

On May 9, 2017, the Virginia Department of Health was notified regarding a patient with suspected rabies. The patient had sustained a dog bite 6 weeks before symptom onset while traveling in India. On May 11, CDC confirmed that the patient was infected with a rabies virus that circulates in dogs in India. Despite aggressive treatment, the patient died, becoming the ninth person exposed to rabies abroad who has died from rabies in the United States since 2008. A total of 250 health care workers were assessed for exposure to the patient, 72 (29%) of whom were advised to initiate postexposure prophylaxis (PEP). The total pharmaceutical cost for PEP (rabies immunoglobulin and rabies vaccine) was approximately $235,000. International travelers should consider a pretravel consultation with travel health specialists; rabies preexposure prophylaxis is warranted for travelers who will be in rabies endemic countries for long durations, in remote areas, or who plan activities that might put them at risk for a rabies exposures.

The potential effect of improved provision of rabies post-exposure prophylaxis in Gavi-eligible countries: a modelling study.

BACKGROUND: Tens of thousands of people die from dog-mediated rabies annually. Deaths can be prevented through post-exposure prophylaxis for people who have been bitten, and the disease eliminated through dog vaccination. Current post-exposure prophylaxis use saves many lives, but availability remains poor in many rabies-endemic countries due to high costs, poor access, and supply. METHODS: We developed epidemiological and economic models to investigate the effect of an investment in post-exposure prophylaxis by Gavi, the Vaccine Alliance. We modelled post-exposure prophylaxis use according to the status quo, with improved access using WHO-recommended intradermal vaccination, with and without rabies immunoglobulin, and with and without dog vaccination. We took the health provider perspective, including only direct costs. FINDINGS: We predict more than 1 million deaths will occur in the 67 rabies-endemic countries considered from 2020 to 2035, under the status quo. Current post-exposure prophylaxis use prevents approximately 56 000 deaths annually. Expanded access to, and free provision of, post-exposure prophylaxis would prevent an additional 489 000 deaths between 2020 and 2035. Under this switch to efficient intradermal post-exposure prophylaxis regimens, total projected vaccine needs remain similar (about 73 million vials) yet 17·4 million more people are vaccinated, making this an extremely cost-effective method, with costs of US$635 per death averted and $33 per disability-adjusted life-years averted. Scaling up dog vaccination programmes could eliminate dog-mediated rabies over this time period; improved post-exposure prophylaxis access remains cost-effective under this scenario, especially in combination with patient risk assessments to reduce unnecessary post-exposure prophylaxis use. INTERPRETATION: Investing in post-exposure vaccines would be an extremely cost-effective intervention that could substantially reduce disease burden and catalyse dog vaccination efforts to eliminate dog-mediated rabies. FUNDING: World Health Organization.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil.

INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.

Rabies risk and use of post-exposure prophylaxis associated with dog bites in Tennessee.

The canine variant of the rabies virus has been eliminated in the United States. Among the public and many healthcare providers, however, dog bites are still associated with risk for rabies transmission. This study examined the risk of rabies in biting dogs and the use of rabies post-exposure prophylaxis (rPEP) for dog bite victims in Tennessee. The study included a retrospective analysis of laboratory testing requisitions for dogs from 2002 to 2016, collection of clinical data on confirmed rabies-positive dogs from 2008 to 2016 and analysis of hospital discharge data for rPEP from 2007 to 2014. Among dogs submitted for rabies testing, those having a recent history of biting were significantly less likely to test positive for rabies than dogs with no reported bite (OR = 0.01; 95% CI [0.003-0.04]). The most common clinical signs reported among rabies-positive dogs were anorexia, dysphagia, ataxia, limb paresis or paralysis, and lethargy; aggressiveness was uncommon. Among hospital patients with an animal-related injury who received rPEP, more than half (52%) presented with dog bites. These data show that laboratory submissions for rabies testing and prescriptions for rPEP do not reflect the epidemiology of rabies in Tennessee. Education and outreach targeting the public and healthcare providers should emphasize the animal species and situations associated with a greater risk for rabies transmission, such as bites from rabies reservoir species or animals exhibiting signs of neurologic disease.

Comparative analysis of Mouse Inoculation Test and Virus Isolation in Cell Culture for rabies diagnosis in animals of Parana, Brazil

Abstract INTRODUCTION: Rabies is an acute zoonotic disease, caused by a rhabdovirus that can affect all mammals, and is commonly transmitted by the bite of a rabid animal. The definitive diagnosis is laboratorial, by the Fluorescent Antibody Test (FAT) as a quick test and Mouse Inoculation Test (MIT) as a confirmatory test (gold standard). Studies conducted over the past three decades indicate that MIT and Virus Isolation in Cell Culture (VICC) can provide the same effectiveness, the latter being considered superior in bioethics and animal welfare. The aim of this study was to compare VICC with MIT, in terms of accuracy, biosafety and occupational health, supply and equipment costs, bioethics and animal welfare, in a Brazilian public health lab. METHODS: We utilized 400 samples of animal neurological tissue to compare the performance of VICC against MIT. The variables analyzed were accuracy, biosafety and occupational health, time spent in performing the tests, supply and equipment costs, bioethics and animal welfare evaluation. RESULTS: Both VICC and MIT had almost the same accuracy (99.8%), although VICC presented fewer risks regarding biosafety and mental health of the technicians, and reduced time between inoculation and obtaining the results (approximately 22 days less). In addition, VICC presented lower supply costs (86.5% less), equipment costs (32.6% less), and the advantage of not using animals. CONCLUSIONS: These results confirm that VICC can replace MIT, offering the same accuracy and better features regarding cost, results, biosafety and occupational health, and bioethics and animal welfare.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

Comparative assessment of seller's staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

BACKGROUND: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller's stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. OBJECTIVE: This study was designed to assess the sensitivity and specificity of the SST in relation to the 'gold standard' dFAT in diagnosis of rabies in Nigeria. METHODS: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. RESULTS: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). CONCLUSION: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

Comparative costs of the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for use in rabies diagnosis in Brazil.

The decision to use laboratory animals rather than in vitro methods is frequently based on the financial costs involved, so the objective of our study was to compare the costs of performing the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for use in rabies diagnosis in Brazil. Based on observations of laboratory routines at the Pasteur Institute, São Paulo, we listed the fixed cost (FC) and variable cost (VC) items necessary to perform both tests. Considering that 200 MITs are equivalent to 350 VICC assays, in terms of facilities and staff-hours needed per month, we calculated, for both tests, the average total cost per sample, the costs of the implementation of the laboratory structure, and the costs of routine use. With regard to absolute values, the total cost was mainly influenced by FC items, as they represented 60% of the cost for the MIT and 86% of the cost for VICC. A sample analysed by the MIT costs around 205% more than one analysed by using VICC. The MIT costs 74% and 406% more than VICC, when implementation costs and routine use per month, respectively, are taken into account. Our results can assist in the resolution of costing disputes that could hinder the replacement of animals for rabies diagnosis in Brazil. The method demonstrated here might also be useful for cost comparisons in other situations where animal use still continues when validated alternatives exist.

Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment.

The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test-a latex agglutination test (LAT)-was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.

Assessment of risk for exposure to bats in sleeping quarters before and during remediation - Kentucky, 2012.

Bats are a reservoir for rabies viruses and have been identified as the most common source of human rabies infections acquired in the United States. The last human rabies fatality from a bat exposure reported in a Kentucky resident occurred in 1996. In July 2012, the Kentucky Department for Public Health (KDPH) was advised of multiple potential bat exposures following efforts to eliminate a bat colony from a volunteer facility. Bats had routinely been sighted in a brick building in eastern Kentucky that had been used as sleeping quarters by an organization that, since 1999, had hosted thousands of children and adults who performed stints of volunteer work over the course of several days. To assess the risk for bat exposure, KDPH and CDC interviewed 257 (94%) of the 273 volunteers and staff members who had slept in the facility in 2012. As a result of that assessment, 48 (19%) persons were identified as potentially exposed, and 16 (33%) of the 48 were recommended to receive rabies postexposure prophylaxis (PEP), including three persons categorized as at high risk and 13 as at moderate risk for exposure. This report highlights the need for guidelines for appropriate remediation of bat infestation and public health investigations of potential mass bat contacts.

Evaluation of an indirect rapid immunohistochemistry test for the differentiation of rabies virus variants.

Cost effective diagnostic tests are needed in rabies virus (RABV) enzootic areas to study the prevalence, distribution, and transmission of rabies virus among reservoir hosts. To reduce the associated costs of acquiring and maintaining specialized laboratory equipment, an indirect rapid immunohistochemistry test (IRIT), for the detection and differentiation of RABV variants, was evaluated by traditional light microscopy. The IRIT utilizes fresh frozen brain touch impressions or cell culture monolayers fixed in buffered formalin, a panel of murine anti-nucleoprotein monoclonal antibodies (mAb-N) and commercially available biotin-labeled goat anti-mouse antibody. In this study, 96 RABV isolates, representing 20 RABV variants previously determined by antigenic typing using a panel of mAb-N and the indirect fluorescent antibody test (IFA), and genetic sequence analysis were characterized by IRIT and the results compared. The IRIT results revealed distinct reactivity patterns associated with current and historical RABV reservoir hosts similar to IFA test and genetic sequence analysis. Evaluation of suspected RABV samples through IRIT does not require specialized equipment and is possible to perform in a field setting. Additionally, commercially available labeled secondary antibodies permit the use of a standard panel of unlabeled primary mAbs, without the need for fluorescence microscopy, and should augment existing attempts at antigenic characterization during canine rabies elimination campaigns in developed and developing countries. These results are useful in studying the epizootiology of rabies and inferring the source of infection when unknown.

An assessment of ONRAB oral rabies vaccine persistence in free-ranging mammal populations in Ontario, Canada.

ONRAB is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB in captive animals, we sought to examine persistence and shedding of ONRAB in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB vaccine baits. Samples were screened for the presence and quantity of ONRAB DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB greater than 10(3)EU/mL of ONRAB DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB in animals is short-lived and the likelihood of horizontal transmission to other organisms is low.

Rabid fox bites and human rabies in a village community in southern India: epidemiological and laboratory investigations, management and follow-up.

Human rabies transmitted from wild animals is rarely reported in endemic countries like India, where nearly 95% deaths occur due to bites from rabid dogs. In this paper, we report an incidence of rabid fox bites in a village in southern part of India involving 18 individuals, including 4 children. All people had category III exposures, including bites on the face and neck. The attacking fox was killed by the forest department and buried immediately. The victims of the fox bite did not receive appropriate and adequate postexposure treatment. Thirteen days after the bite, one of the bite victims developed typical symptoms of furious rabies and died 2 days later in a local hospital. His brain tissue, obtained at autopsy, was strongly positive for rabies by fluorescent antibody technique (FAT) and virus isolation. Panic prevailed in the community and the rest of the 17 cases were referred to our institute for advice and further management. Only 35% of them had protective levels of rabies virus neutralizing antibodies (RVNA). All of the patients were administered with an 8-site intradermal regimen with purified chick embryo cell (PCEC) vaccine and were followed up regularly. All of them developed adequate titers (>0.5 IU/mL) of RVNA 7 days later. They were under regular follow-up and after nearly 2 years none have developed rabies. The partial Nucleoprotein (N) gene sequencing of the virus isolate from the patient who died of rabies had close homology with species I (prototype rabies) sequences available in GenBank and our own past isolates from dogs and humans, thus confirming that virus spillover from wildlife to domestic dogs continues to occur. This episode should prompt health authorities to focus more attention on training rural medical practitioners in state-of-the-art modern prophylactic measures.