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.

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.

New isolations of the rabies-related Mokola virus from South Africa.

BACKGROUND: Mokola virus (MOKV) is a rabies-related lyssavirus and appears to be exclusive to the African continent. Only 24 cases of MOKV, which includes two human cases, have been reported since its identification in 1968. MOKV has an unknown reservoir host and current commercial vaccines do not confer protection against MOKV. RESULTS: We describe three new isolations of MOKV from domestic cats in South Africa. Two cases were retrospectively identified from 2012 and an additional one in 2014. CONCLUSIONS: These cases emphasize the generally poor surveillance for rabies-related lyssaviruses and our inadequate comprehension of the epidemiology and ecology of Mokola lyssavirus per se.

Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody.

Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model.

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.

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.

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.

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.

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.

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.

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.

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.

Detection of lyssavirus antigen and antibody levels among apparently healthy and suspected rabid dogs in South-Eastern Nigeria.

OBJECTIVES: Domestic dogs are the main reservoir of rabies virus (RABV) infection in Nigeria, thus surveillance of rabies in dog populations is crucial in order to understand the patterns of spread of infection and ultimately devise an appropriate rabies control strategy. This study determined the presence of lyssavirus antigen in brain tissues and anti-rabies antibodies in sera of apparently healthy and suspected-rabid dogs slaughtered for human consumption at local markets in South-Eastern Nigeria. RESULTS: Our findings demonstrated that 8.3% (n = 23) of brain tissues were lyssavirus positive and 2.5% (n = 25) of sera had rabies antibody levels as percentage blocking of 70% and above correlating with a cut-off value ≥ 0.5 IU/mL in the fluorescent antibody neutralization test. There was an inverse correlation between lyssavirus positivity and rabies antibody levels confirming that infected individuals most often do not develop virus neutralizing antibodies to the disease. The low percentage of rabies antibodies in this dog population suggests a susceptible population at high risk to RABV infection. These findings highlight a huge challenge to national rabies programs and subsequent elimination of the disease from Nigeria, considering that majority of dogs are confined to rural communal areas, where parenteral dog vaccination is not routinely undertaken.

Spatiotemporal epidemiology of rabies at an interface between domestic dogs and wildlife in South Africa.

We characterized the spatiotemporal epidemiology of rabies from January 2009 through March 2014 across the interface between a wildlife reserve and communal livestock farming area in South Africa. Brain tissue from 344 animals of 28 different species were tested for lyssavirus antigen. Of these, 146 (42.4%) samples tested positive, of which 141 (96.6%) came from dogs. Brain samples of dogs were more likely to test positive for lyssavirus antigen if they were found and destroyed in the reserve, compared to samples originating from dogs outside the reserve (65.3% vs. 45.5%; odds ratio (OR) = 2.26, 95% confidence interval (CI) = 1.27-4.03), despite rabies surveillance outside the reserve being targeted to dogs that have a higher index of suspicion due to clinical or epidemiological evidence of infection. In the reserve, dogs were more likely to test positive for rabies if they were shot further from villages (OR = 1.42, 95% CI 1.18-1.71) and closer to water points (OR = 0.41, 95% CI 0.21-0.81). Our results provide a basis for refinement of existing surveillance and control programs to mitigate the threat of spillover of rabies to wildlife populations.

Rabies of canid biotype in wild dog (Lycaon pictus) and spotted hyaena (Crocuta crocuta) in Madikwe Game Reserve, South Africa in 2014-2015: Diagnosis, possible origins and implications for control.

Both domestic and wild carnivore species are commonly diagnosed with rabies virus (RABV) infection in South Africa. Although the majority of confirmed rabies cases in wild carnivore species are reported from the yellow mongoose (Cynictis penicillata), the rest are from other wild carnivores including the highly endangered wild dog (Lycaon pictus). Lyssavirus infection was confirmed in two wild dogs and a spotted hyaena (Crocuta crocuta) in the Madikwe Game Reserve, North West province in South Africa, in 2014 and 2015, using a direct fluorescent antibody test and immunohistochemistry. There had been no new wild dog introductions to the Madikwe Game Reserve for many years and the wild dogs were last vaccinated against rabies approximately 11 years prior to the incident. The first euthanised wild dog was the last surviving of a break-away pack of 6, and the second was the last of a larger pack of 18, the rest of which died with no carcasses being found or carcasses too decomposed for sampling. Subsequent antigenic typing of the lyssaviruses indicated that they were canid RABVs. The RABVs originating from 22 wild carnivore species, 7 dogs, and a caprine, mostly from the North West province, were genetically characterised by targeting a partial region of the nucleoprotein gene. The nucleotide sequence analyses of these viruses and two previously characterised RABVs confirmed that the outbreak viruses were also canid rabies, phylogenetically clustering with virus isolates originating from black-backed jackals recovered between 2012 and 2015 from the North West province, and domestic dogs from neighbouring communal areas. The source(s) of the mortalities and possible reservoir host(s) for the virus could only be speculated upon from data on specific predator numbers, movements and behaviour, kills, park management and the changing environmental ecology, which were monitored closely in Madikwe over several years. The most likely rabies sources were from boundary fence contacts between wild carnivores within the park, with domestic dogs or cats and/or naturally occurring wild carnivores outside the park. The associated risk of zoonotic infection and threat to important and endangered predators may be mitigated through regional rabies control primarily in domestic dogs and cats, as well as by preventative vaccination of at-risk park employees and their pets. The importance of ongoing prophylactic rabies protection by regular vaccination of highly endangered wildlife carnivores and the submission of carcasses for rabies diagnosis of any wild or domestic animals behaving uncharacteristically or found dead is emphasised.

Enhanced diagnosis of rabies and molecular evidence for the transboundary spread of the disease in Mozambique.

Rabies is a neglected zoonotic disease with veterinary and public health significance, particularly in Africa and Asia. The current knowledge of the epidemiology of rabies in Mozambique is limited because of inadequate sample submission, constrained diagnostic capabilities and a lack of molecular epidemiological research. We wanted to consider the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) for rabies diagnosis at the diagnostic laboratory of the Central Veterinary Laboratory (CVL), Directorate of Animal Science, Maputo, Mozambique. Towards this aim, as a training exercise at the World Organisation for Animal Health (OIE) Rabies Reference Laboratory in South Africa, we performed the DRIT on 29 rabies samples from across Mozambique. With the use of the DRIT, we found 15 of the 29 samples (52%) to be negative. The DRIT-negative samples were retested by DFA at the OIE Rabies Reference Laboratory, as well as with an established real-time Polymerase chain reaction, confirming the DRIT-negative results. The DRIT-positive results (14/29) were retested with the DFA and subsequently amplified, sequenced and subjected to phylogenetic analyses, confirming the presence of rabies RNA. Molecular epidemiological analyses that included viruses from neighbouring countries suggested that rabies cycles within Mozambique might be implicated in multiple instances of cross-border transmission. In this regard, our study has provided new insights that should be helpful in informing the next steps required to better diagnose, control and hopefully eliminate rabies in Mozambique.

Evaluation of Six Commercially Available Rapid Immunochromatographic Tests for the Diagnosis of Rabies in Brain Material.

Rabies is a neglected zoonotic disease that causes an estimated 60,000 human deaths annually. The main burden lies on developing countries in Asia and Africa, where surveillance and disease detection is hampered by absence of adequate laboratory facilities and/or the difficulties of submitting samples from remote areas to laboratories. Under these conditions, easy-to-use tests such as immunochromatographic assays, i.e. lateral flow devices (LFD), may increase surveillance and improve control efforts. Several LFDs for rabies diagnosis are available but, except for one, there are no data regarding their performance. Therefore, we compared six commercially available LFDs for diagnostic and analytical sensitivity, as well as their specificity and their diagnostic agreement with standard rabies diagnostic techniques using different sample sets, including experimentally infected animals and several sets of field samples. Using field samples the sensitivities ranged between 0% up to 100% depending on the LFD and the samples, while for experimentally infected animals the maximum sensitivity was 32%. Positive results in LFD could be further validated using RT-qPCR and sequencing. In summary, in our study none of the tests investigated proved to be satisfactory, although the results somewhat contradict previous studies, indicating batch to batch variation. The high number of false negative results reiterates the necessity to perform a proper test validation before being marketed and used in the field. In this respect, marketing authorization and batch release control could secure a sufficient quality for these alternative tests, which could then fulfil their potential.

Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa.

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.