Rabies in Cats-An Emerging Public Health Issue.

Human rabies cases today are predominantly associated with infection from rabid domestic dogs. Unlike dogs, a common global reservoir species that perpetuates rabies viruses (RABV) within their populations, domestic cats are much less frequently reported or vaccinated. Epidemiologically, cats are important vectors of lyssaviruses but are not viral reservoirs. Typically, cats are incidental hosts only, infected with the predominant lyssavirus in their geographic locale. Human cases associated with rabid cats have occurred in Africa, Asia, Europe and throughout the Americas. As adept, solitary hunters, wild and domestic felids are at risk of lyssavirus infection based upon interactions with infected prey, such as bats, or from transmission by other mesocarnivores, such as rabid dogs, foxes, jackals, raccoons, and skunks. Current veterinary vaccines provide safe and effective immunity in cats against phylogroup I lyssaviruses, such as RABV, but not against divergent lyssaviruses in phylogroups II-IV. With the focus upon the global elimination of canine rabies, the emergence of rabies in cats represents a concerning trend. Clearly, education about the occurrence of rabies in cats needs to be improved, as well as the routine vaccination of cats to reduce the associated risks to public health, agriculture, and conservation biology from a One Health perspective.

Patterns of Animal Rabies Prevalence in Northern South Africa between 1998 and 2022.

Rabies is endemic in South Africa and rabies cycles are maintained in both domestic and wildlife species. The significant number of canine rabies cases reported by the World Organization for Animal Health Reference Laboratory for Rabies at Onderstepoort suggests the need for increased research and mass dog vaccinations on specific targeted foci in the country. This study aimed to investigate the spatiotemporal distribution of animal rabies cases from 1998 to 2017 in northern South Africa and environmental factors associated with highly enzootic municipalities. A descriptive analysis was used to investigate temporal patterns. The Getis-Ord Gi statistical tool was used to exhibit low and high clusters. Logistic regression was used to examine the association between the predictor variables and highly enzootic municipalities. A total of 9580 specimens were submitted for rabies diagnosis between 1998 and 2022. The highest positive case rates were from companion animals (1733 cases, 59.71%), followed by livestock (635 cases, 21.88%) and wildlife (621 cases, 21.39%). Rabies cases were reported throughout the year, with the majority occurring in the mid-dry season. Hot spots were frequently in the northern and eastern parts of Limpopo and Mpumalanga. Thicket bush and grassland were associated with rabies between 1998 and 2002. However, between 2008 and 2012, cultivated commercial crops and waterbodies were associated with rabies occurrence. In the last period, plantations and woodlands were associated with animal rabies. Of the total number of municipalities, five consistently and repeatedly had the highest rabies prevalence rates. These findings suggest that authorities should prioritize resources for those municipalities for rabies elimination and management.

Serological evidence of virus infection in Eidolon helvum fruit bats: implications for bushmeat consumption in Nigeria.

Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.

Molecular Characterization of Lyssaviruses Originating from Domestic and Wild Cats Provides an Insight on the Diversity of Lyssaviruses and a Risk of Rabies Transmission to Other Susceptible Mammals and Humans in South Africa.

Rabies is one of the most significant public and veterinary health problems, causing approximately 59,000 human deaths annually in the developing countries of Asia and Africa. The aetiologic agent, a viral species of the Lyssavirus genus, is highly neurotropic and has a wide host range, including terrestrial mammals and several Chiropteran species. The Lyssavirus mokola (MOKV) was first isolated in the late 1960s from organ pools of shrews (Crocidura flavescens manni) in the Mokola forest (Nigeria). To date, at least 30 MOKV isolations have been confirmed, all exclusively from Africa, with 73% from southern Africa. There is limited knowledge about the epidemiology of MOKV, and the reservoir host species is unknown. Here, we report on the molecular characterization of rabies viruses originating from both domestic and African wild cats. A partial region of the lyssavirus genome, encoding the nucleoprotein, was amplified and sequenced. Nucleotide sequence analysis demonstrated that 98% of cats were infected with both the canid and mongoose rabies virus variants, as well as a rare lyssavirus, Lyssavirus mokola, from a domestic cat from Eswatini. Furthermore, the nucleotide sequence divergence between the recently identified MOKV isolate and the historical Lyssavirus mokola isolates ranged from 6.8% to 8.3%. This study further highlights the association between the potential host species of Lyssavirus mokola and the domestic cat as an incidental host, and the important role cats may play in rabies transmission dynamics in the country. Therefore, continuous vaccination of domestic cats against rabies is crucial, even after the elimination of dog-mediated rabies, as spillover related to sylvatic rabies cycles is likely to occur.

A Case Report on a Human Bite Contact with a Rabid Honey Badger Mellivora capensis (Kromdraai Area, Cradle of Humankind, South Africa).

In South Africa, rabies cycles are sustained by both domestic and wildlife host species. Despite the fact that the majority of human rabies cases are associated with dog bite exposures, wildlife species can potentially transmit rabies virus (RABV) infection to humans. In July 2021, a honey badger (Mellivora capensis) from the Kromdraai area (Gauteng Province) bit a dog on a small farm. The following day the same honey badger attacked three adults in the area, with one of the victims requiring hospitalization for management of her injuries. The honey badger was subsequently shot and the carcass submitted to the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR) for RABV diagnosis. A positive rabies diagnosis was confirmed and phylogenetic analysis of the amplified glycoprotein gene of the rabies virus demonstrated the virus to be of dog origin.

Diagnostic sensitivity and specificity of immunohistochemistry for the detection of rabies virus in domestic and wild animals in South Africa.

We estimated the diagnostic sensitivity (DSe) and specificity (DSp) of an immunohistochemistry (IHC) protocol compared to the direct fluorescent antibody test (DFAT), which is the gold standard test for rabies diagnosis. We obtained brain samples from 199 domestic and wild animal cases (100 DFAT-negative, 99 DFAT-positive), by convenience sampling from 2 government-accredited rabies virus (RABV) testing laboratories in South Africa, between February 2015 and August 2017. Tissues that had been stored at 4-8°C for several days to weeks at the 2 accredited laboratories were formalin-fixed and paraffin-embedded. Nighty-eight cases tested IHC-positive using a polyclonal anti-RABV nucleoprotein antibody and a polymer detection system. The overall DSe and DSp for the RABV IHC test were 98% (95% CI: 93-100%) and 99% (95% CI: 95-100%), respectively. Domestic dogs accounted for 41 of 98 RABV IHC-positive cases, with the remainder in 4 domestic cats, 25 livestock, and 28 wildlife. Herpestidae species, including 7 meerkats and 9 other mongoose species, were the most frequently infected wild carnivores, followed by 11 jackals. Three cases in domestic dogs had discordant test results; 2 cases were IHC-/DFAT+ and 1 case was IHC+/DFAT-. Considering the implications of a false-negative rabies diagnosis, participating in regular inter-laboratory comparisons is vital, and a secondary or confirmatory method, such as IHC, should be performed on all submitted specimens, particularly negative cases with human contact history.

Public Health Awareness on Bat Rabies among Bat Handlers and Persons Residing near Bat Roosts in Makurdi, Nigeria.

Rabies is a neglected disease endemic in Asia and Africa but is still a significant public and veterinary health threat. Whilst a key delicacy for the local diet, bats are a natural reservoir host for many viral zoonotic agents including lyssaviruses, the causative agent of rabies. Studies on knowledge and practices linked to the disease will help to identify gaps and define preventive strategies that may subsequently result in a reduction and the potential elimination of human rabies. In order to assess the public health awareness of bat rabies among specific population groups in Makurdi (Nigeria), structured questionnaires (n = 154) were administered by face-to-face interviews to bat handlers and persons residing near bat roost sites. A total of 59.7% of the respondents were persons residing near bat roost sites, 13% were bat hunters, 25.3% were bat meat consumers and 1.9% were university researchers. Only 6.5% of respondents reported using some form of personal protective equipment (PPE) ranging from hand gloves, face/nose masks and protective boots to lab coats/coveralls while handling bats, whilst the majority (93.5%) did not use any form of PPE. With a mean knowledge score of 8.34 out of a possible 12 points, 50.6% of respondents had good knowledge of bats and their disease-carrying potential, 39.6% had fair knowledge, while 9.7% had poor knowledge. Log linear models showed significant associations between knowledge score and level of education, as well as knowledge score and occupation. The latter highlights the requirement to enhance public education among bat handlers and persons residing near bat roosts on the need to protect themselves better, while handling bats particularly during processing of bats for food and on steps to take when exposed to bites from bats.

Rabies Virus Seroprevalence among Dogs in Limpopo National Park and the Phylogenetic Analyses of Rabies Viruses in Mozambique.

Rabies is considered a neglected disease among many developing Asian and African countries, including Mozambique, where its re-emergence is often attributed to low dog parenteral vaccination coverage. The objectives of this study were two-fold: (1) to assess the level of antibodies against rabies virus in dogs (n = 418) in Limpopo National Park (LNP), and (2) to genetically characterise selected rabies viruses from brain tissue samples collected in 2017 and 2018. To meet the first objective, we used the BioProTM Rabies blocking ELISA antibody kit, and the results were expressed as the percentage of blocking (%PB). Dog sera with PB ≥ 40% were considered positive for antibodies to rabies virus, whereas sera with PB < 40% were negative. Just under ninety percent (89.2%; n = 373) of dogs were seronegative, and the rest (10.8%; n = 45) had detectable levels of rabies virus-specific antibodies. All eight brain tissue samples were positive for rabies virus antigen using a direct fluorescent antibody test and amplified in a quantitative real-time PCR, but only five (n = 4 from dogs and n = 1 from a cat) were amplified in a conventional reverse-transcription PCR targeting partial regions of the nucleoprotein (N) and the glycoprotein (G) genes. All samples were successfully sequenced. Phylogenetically, the rabies viruses were all of dog origin and were very closely related to each other (Africa 1b rabies virus lineage). Furthermore, the sequences had a common progenitor with other rabies viruses from southern Africa, confirming the transboundary nature of rabies and the pivotal role of dogs in maintaining rabies cycles. The study demonstrates the principal application of the BioProTM rabies ELISA antibody for the detection of anti-lyssavirus-specific antibodies in the serum samples of dogs, and most importantly, it highlights the low levels of antibodies against rabies virus in this dog population.

Spatio-temporal epidemiology of animal and human rabies in northern South Africa between 1998 and 2017.

BACKGROUND: Rabies is a fatal zoonotic disease that is maintained in domestic dogs and wildlife populations in the Republic of South Africa. A retrospective study was conducted to improve understanding of the dynamics of rabies in humans, domestic dogs, and wildlife species, in relation to the ecology for three northern provinces of South Africa (Limpopo, Mpumalanga, and North-West) between 1998 and 2017. METHODS: A descriptive epidemiology study was conducted for human and animal rabies. Dog rabies cases were analyzed using spatio-temporal scan statistics. The reproductive number (Rt) was estimated for the identified disease clusters. A phylogenetic tree was constructed based on the genome sequences of rabies viruses isolated from dogs, jackals, and an African civet, and Bayesian evolutionary analysis using a strict time clock model. Several ecological and socio-economic variables associated with dog rabies were modeled using univariate analyses with zero-inflated negative binomial regression and multivariable spatial analyses using the integrated nested Laplace approximation for two time periods: 1998-2002 and 2008-2012. RESULTS: Human rabies cases increased in 2006 following an increase in dog rabies cases; however, the human cases declined in the next year while dog rabies cases fluctuated. Ten disease clusters of dog rabies were identified, and utilizing the phylogenetic tree, the dynamics of animal rabies over 20 years was elucidated. In 2006, a virus strain that re-emerged in eastern Limpopo Province caused the large and persistent dog rabies outbreaks in Limpopo and Mpumalanga Provinces. Several clusters included a rabies virus variant maintained in jackals in Limpopo Province, and the other variant in dogs widely distributed. The widely distributed variant maintained in jackal populations in North-West Province caused an outbreak in dogs in 2014. The Rt was high when the disease clusters were associated with either multiple virus strains or multiple animal species. High-risk areas included Limpopo and Mpumalanga Provinces characterized by woodlands and high temperatures and precipitation. CONCLUSION: Canine rabies was maintained mainly in dog populations but was also associated with jackal species. Rural communities in Limpopo and Mpumalanga Provinces were at high risk of canine rabies originating from dogs.

Epidemiological Interface of Sylvatic and Dog Rabies in the North West Province of South Africa.

Rabies is a viral zoonosis that causes an estimated 59,000 preventable human fatalities every year. While more than 120 countries remain endemic for dog-mediated rabies, the burden is the highest in Africa and Asia where 99% of human rabies cases are caused by domestic dogs. One such rabies-endemic country is South Africa where an estimated 42 preventable human deaths occur every year. Although canine rabies had been well described for most of the provinces in South Africa, the epidemiology of rabies within the North West Province had not been well defined prior to this investigation. As such, the aim of this study was to use nucleotide sequence analyses to characterise the extant molecular epidemiology of rabies in the North West Province of South Africa-with specific focus on the interface between dogs and sylvatic species. To this end, Rabies lyssavirus isolates originating from the North West Province were subjected to molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology on two distinct gene regions, viz. the G-L intergenic region and partial nucleoprotein gene. Our results provided strong evidence in support of an endemic cycle of canine rabies in the East of the province, and three independent endemic cycles of sylvatic rabies spread throughout the province. Furthermore, evidence of specific events of virus spill-over between co-habiting sylvatic species and domestic dogs was found. These results suggest that the elimination of canine-mediated rabies from the province will rely not only on eliminating the disease from the dog populations, but also from the co-habiting sylvatic populations using oral rabies vaccination campaigns.

Knowledge, attitudes, practices (KAP) and control of rabies among community households and health practitioners at the human-wildlife interface in Limpopo National Park, Massingir District, Mozambique.

BACKGROUND: Rabies is a viral zoonotic disease that kills more than 26,000 people each year in Africa. In Mozambique, poverty and inadequate surveillance result in gross underreporting and ineffective control of the disease in animals and people. Little is known of the role of human attitudes and behaviour in prevention or control of rabies, thus this study was undertaken to assess the knowledge, attitudes and practices amongst selected households and health practitioners in one affected area, the Limpopo National Park (LNP), Massingir district. METHODOLOGY: A cross-sectional study was conducted among 233 households in eight villages in LNP and among 42 health practitioners from eight health facilities in Massingir district between 2016 and 2018. Consenting household representatives aged 18 years or over were purposively selected. A KAP survey was administered to obtain information on dog ownership and knowledge of rabies, host species affected, modes of transmission, symptoms, recommended treatment and preventive methods. Similar to household study participants, health practitioners were purposively selected and completed the questionnaire during the investigators' visit. The questionnaire sought information on knowledge of rabies, management of bite wounds, vaccination sites and schedules of pre- and post-exposure prophylaxis. Descriptive and inferential data analyses were performed using SPSS software version 18.0. RESULTS: Approximately twenty per cent (18.9%; 95% CI: 14.3-24.3) and 13.3% (95% CI: 9.4-18.1) of households had good knowledge and practices of rabies, respectively. For health practitioners, only 16.7% (95% CI: 7.5-31.9) had good knowledge, whilst 33.3% (95% CI: 20.0-49.7) adopted adequate attitudes/practices towards the disease. CONCLUSIONS/SIGNIFICANCE: In conclusion, both households and health practitioners displayed poor levels of knowledge and adopted bad attitudes and practices towards rabies. The former, had more gaps in their attitudes and practices towards the disease. Village location and education level (P < .05) and similarly, sex and occupation, were found to be statistically associated with good knowledge of rabies among households as compared to HPs. Overall, a lack of community-based education and professional retraining courses contribute significantly to poor awareness of rabies in the LNP of Mozambique. Enhancing public health knowledge should consequently reduce dog-mediated human rabies deaths in this country.

Serum Neutralization Profiles of Straw-Colored Fruit Bats (Eidolon helvum) in Makurdi (Nigeria), against Four Lineages of Lagos Bat Lyssavirus.

Lagos bat lyssavirus (LBV) comprising four lineages (A, B, C and D) can potentially cause the fatal disease rabies. Although LBV-B was initially isolated in Nigeria in 1956, there is no information on LBV lineages circulating in Nigeria. This study was undertaken for the first time to measure the neutralizing antibodies against four lineages of LBVs in straw-colored fruit bats (Eidolon helvum) in Makurdi, Nigeria. Serum samples (n = 180) collected during two periods (November 2017-March 2018 and November 2018-March 2019) from terminally bled bats captured for human consumption were tested using a modified fluorescent antibody virus neutralization (mFAVN) assay. A high proportion of bat sera (74%) neutralized at least one lineage of LBV (with reciprocal titers from 9 to >420.89) and most of them neutralized LBV-A (63%), followed by LBV-D (49%), LBV-C (45%) and LBV-B (24%). The majority of positive sera (75%, n = 100) neutralized multiple LBV lineages while the remaining 25% (n = 33) neutralized only a single lineage, i.e., LBV-A (n = 23), LBV-D (n = 8) and LBV-C (n = 2). None exclusively neutralized LBV-B. The results suggest that exposure to LBV is common in E. helvum and that LBV-A (but not LBV-B) is likely to be circulating in this region of Nigeria.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection.

We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

Role of Oral Rabies Vaccines in the Elimination of Dog-Mediated Human Rabies Deaths.

Domestic dogs are responsible for nearly all the ¼59,000 global human rabies deaths that occur annually. Numerous control measures have been successful at eliminating dog-mediated human rabies deaths in upper-income countries, including dog population management, parenteral dog vaccination programs, access to human rabies vaccines, and education programs for bite prevention and wound treatment. Implementing these techniques in resource-poor settings can be challenging; perhaps the greatest challenge is maintaining adequate herd immunity in free-roaming dog populations. Oral rabies vaccines have been a cornerstone in rabies virus elimination from wildlife populations; however, oral vaccines have never been effectively used to control dog-mediated rabies. Here, we convey the perspectives of the World Organisation for Animal Health Rabies Reference Laboratory Directors, the World Organisation for Animal Health expert committee on dog rabies control, and World Health Organization regarding the role of oral vaccines for dogs. We also issue recommendations for overcoming hesitations to expedited field use of appropriate oral vaccines.

Lyssaviruses in Insectivorous Bats, South Africa, 2003-2018.

We detected 3 lyssaviruses in insectivorous bats sampled in South Africa during 2003-2018. We used phylogenetic analysis to identify Duvenhage lyssavirus and a potentially new lyssavirus, provisionally named Matlo bat lyssavirus, that is related to West Caucasian bat virus. These new detections highlight that much about lyssaviruses remains unknown.

Complete Coding Sequences of 23 South African Domestic and Wildlife Rabies Viruses.

South African rabies viruses originating from dogs and jackals (canid viruses) are closely related and highlight cross-species transmission events between the two canine species. Rabies due to the canid lyssavirus variant is a significant public health matter in this country. The complete coding sequences of 23 canid lyssaviruses from South Africa are reported here.

Rabies in the African Civet: An Incidental Host for Lyssaviruses?

In South Africa, canid rabies virus (RABV) infection is maintained in domestic and wildlife species. The identification of rabies in African civets raised the question of whether this wildlife carnivore is a potential reservoir host of RABVs of direct and ancestral dog origin (dog-maintained and dog-derived origins) with an independent cycle of transmission. Genetic analyses of African civet nucleoprotein sequences for 23 African civet RABVs and historically published sequences demonstrated that RABVs from African civets have two origins related to dog and mongoose rabies enzootics. The data support observations of the interaction of civets with domestic dogs and wildlife mongooses, mostly in Northern South Africa and North-East Zimbabwe. Within each host species clade, African civet RABVs group exclusively together, implying intra-species virus transfer occurs readily. The canid RABV clade appears to support virus transfer more readily between hosts than mongoose RABVs. Furthermore, these data probably indicate short transmission chains with conspecifics that may be related to transient rabies maintenance in African civets. Hence, it is important to continue monitoring the emergence of lyssaviruses in this host. Observations from this study are supported by ongoing and independent similar cases, in which bat-eared foxes and black-backed jackal species maintain independent rabies cycles of what were once dog-maintained RABVs.

Rabies Vaccination of 6-Week-Old Puppies Born to Immunized Mothers: A Randomized Controlled Trial in a High-Mortality Population of Owned, Free-Roaming Dogs.

To achieve global elimination of human rabies from dogs by 2030, evidence-based strategies for effective dog vaccination are needed. Current guidelines recommend inclusion of dogs younger than 3 months in mass rabies vaccination campaigns, although available vaccines are only recommended for use by manufacturers in older dogs, ostensibly due to concerns over interference of maternally-acquired immunity with immune response to the vaccine. Adverse effects of vaccination in this age group of dogs have also not been adequately assessed under field conditions. In a single-site, owner-blinded, randomized, placebo-controlled trial in puppies born to mothers vaccinated within the previous 18 months in a high-mortality population of owned, free-roaming dogs in South Africa, we assessed immunogenicity and effect on survival to all causes of mortality of a single dose of rabies vaccine administered at 6 weeks of age. We found that puppies did not have appreciable levels of maternally-derived antibodies at 6 weeks of age (geometric mean titer 0.065 IU/mL, 95% CI 0.061-0.069; n = 346), and that 88% (95% CI 80.7-93.3) of puppies vaccinated at 6 weeks had titers ≥0.5 IU/mL 21 days later (n = 117). Although the average effect of vaccination on survival was not statistically significant (hazard ratio [HR] 1.35, 95% CI 0.83-2.18), this effect was modified by sex (p = 0.02), with the HR in females 3.09 (95% CI 1.24-7.69) and the HR in males 0.79 (95% CI 0.41-1.53). We speculate that this effect is related to the observed survival advantage that females had over males in the unvaccinated group (HR 0.27; 95% CI 0.11-0.70), with vaccination eroding this advantage through as-yet-unknown mechanisms.

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.

Molecular Detection of Rabies Lyssaviruses from Dogs in Southeastern Nigeria: Evidence of TransboundaryTransmission of Rabies in West Africa.

Despite being the first country to register confirmed cases of Mokola and Lagos bat lyssaviruses (two very distant lyssaviruses), knowledge gaps, particularly on the molecular epidemiology of lyssaviruses, still exist in Nigeria. A total of 278 specimens were collected from dogs in southeastern Nigeria between October 2015 and July 2016, and 23 (8.3%) of these tested positive for lyssaviruses with the direct fluorescent antibody test (DFA). The lyssaviruses were genetically characterized by amplifying the highly conserved nucleoprotein (N) gene of the rabies lyssaviruses (RABVs) of the viral genome. Phylogenetic analyses of the nucleotide sequences showed that all the RABV sequences in this study were of the Africa-2 lineage. Our results demonstrated that transboundary transmission of rabies lyssavirus is a key event, given that one of the RABV sequences (MN196576) clustered with rabies variants from neighboring Niger Republic. Furthermore, three RABVs from dogs from Anambra State clustered separately forming a novel and distinct group. Our results demonstrated that transboundary transmission of RABLVs is a key driver in the spread of rabies in West Africa. In order for the successful control of this zoonotic disease, a multinational stepwise surveillance and elimination of rabies in Africa by 2030 is probably the solution for regional elimination.