Fluorescent Antibody Virus Neutralization Test Research Articles

Effect of Rabies Booster Vaccination on Antibody Levels in African Wild Dogs (Lycaon pictus).

Rabies is a highly virulent viral disease that has been associated with large-scale population declines of the endangered African wild dog (Lycaon pictus). Rabies vaccination may be a valuable conservation tool in this species, but studies indicate that a single dose does not always confer protective immunity. We examined 47 serum samples from 22 captive African wild dogs (sampled opportunistically for other purposes) to assess whether serum antibody levels after vaccination correlated with the number of doses received and whether other factors affected outcomes. Results of the fluorescent antibody virus neutralization test showed that median antibody titers were 0.085 IU/mL prevaccination, 0.660 IU/mL after a single vaccination, and 22.150 IU/mL after a booster vaccination. Antibody titers above 0.5 IU/mL, internationally accepted as the threshold for seroconversion, were found in none of the samples taken prevaccination, 66.67% of samples taken after primary vaccination, and 90.90% of samples collected after booster vaccination. This study illustrates the probable protective benefit a rabies booster vaccination may provide in African wild dogs and serves as a basis for future research to improve vaccination protocols contributing to the conservation of this endangered species.

Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Evaluation of an iELISA for detection and quantification of rabies antibodies in domestic dog sera

Many rabies endemic-countries have recognized rabies as a public health problem that can be eliminated. As a result, some countries have started implementing small-scale vaccination programs with the aim of scaling them up. Post-vaccination serological monitoring is crucial to assess the efficacy of these programs. The recommended serological tests, the rapid fluorescent focus inhibition test, and the fluorescent antibody virus neutralization (FAVN) are accurate; however, the procedures require considerable expertise and must be carried out in high containment facilities, which are often not available in rabies endemic countries. Given these constraints, enzyme linked immunosorbent assays (ELISAs) have been considered as alternative methods to neutralization tests. This is the first study to evaluate, under field conditions, the performance of the commercial rabies indirect-ELISA (iELISA), the PlateliaTM Rabies II kit ad usum Veterinarium kit, using sera from domestic dogs. Serum samples were collected from two groups of community dogs in northern Tanzania: i) dogs with no history of vaccination against rabies (n = 100) and ii) dogs vaccinated with the Nobivac Canine Rabies® vaccine (n = 101) four weeks previously. When compared to the gold standard FAVN test, the iELISA was found to be 99% specific and 98% sensitive and there was a significant correlation between the two tests (p < 0.001, r = 0.92). Given these findings, we conclude that the PlateliaTM Rabies II kit ad usum Veterinarium can be considered a valuable tool for the rapid assessment of vaccination status of animals in vaccination programs.

A simplified method for measuring neutralising antibodies against rabies virus

Rabies virus (RABV) causes a fatal encephalitis that can be prevented through timely vaccination. The levels of virus neutralising antibodies against rabies virus induced by vaccination can be measured using the fluorescent antibody virus neutralisation (FAVN) test. Following incubation of live virus with sera, this method involves the fixation of cell monolayers and staining of rabies virus-specific antigen using fluorescein isothiocyanate (FITC) -conjugated antibody to enable visualisation of rabies virus antigen using a fluorescence microscope. To simplify this procedure, a fluorescent recombinant rabies virus was constructed using reverse genetics by inserting the gene for the mCherry fluorescent protein in front of the ribonucleoprotein gene of the SAD B-19 genome and replacing its glycoprotein with that of the Challenge Virus Standard (CVS)-11 RABV strain to ensure antigenic authenticity with the FAVN. This new recombinant virus (termed mCCCG) expressed the mCherry protein to high levels enabling direct observation of infected cells. In vitro growth kinetics of mCCCG were indistinguishable from that of CVS-11. The stability of the recombinant virus was assessed by sequencing several passages of the rescued virus and only minor changes were detected. Comparative assessment of the virus neutralisation test using mCherry producing virus (NTmCV) against the FAVN demonstrated that test results were equivalent to each other; therefore, mCCCG can be used as an alternative to CVS-11 for measuring antibody titres against the rabies virus. The use of NTmCV removes the need for expensive antibody conjugates and significantly reduces assay time. This would be particularly beneficial for RABV serological assessment in resource limited settings. Moreover, the reading of the plates can be automatically using a cell imaging reader.

Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters

The World Health Organization protocol for rabies post-exposure prophylaxis (PEP) recommends extensive wound washing, immediate vaccination, and administration of rabies immunoglobulin (RIG) in severe category III exposures. Some studies have shown that RIG can interfere with rabies vaccine immunogenicity to some extent. We investigated the interference of RIG on a next generation highly purified Vero cell rabies vaccine candidate (PVRV-NG) versus standard-of-care vaccines in a previously described hamster model. The interference of either human (h) or equine (e) RIG on the immune response elicited by PVRV-NG, Verorab® (purified Vero cell rabies vaccine, PVRV), and Imovax® Rabies (human diploid cell rabies vaccine; HDCV) was evaluated using the 4-dose Essen PEP regimen. The anti-rabies seroneutralizing titers and specific serum IgM titers were measured by fluorescent antibody virus neutralization test and enzyme-linked immunosorbent assay, respectively, for the vaccines administered with or without RIG. The RIG interference on PVRV-NG, observed transiently at Day 7, was similar to that on PVRV and tended to be lower than that on HDCV using both read-outs. In summary, the results generated in the hamster model showed that RIG induced similar or less interference on PVRV-NG than the standard-of-care vaccines.

Optimization of rabies (Rhabdoviridae: Lyssavirus) dog vaccination schedule using a mathematical model

Most cases of human rabies are caused by dog (Canis lupus familiaris) bites. Therefore, the implementation of vaccination programs of these animals is one of the urgent tasks.The work aims to identify the factors influencing the production of antirabies virus-neutralizing antibodies (VNAs) in vaccinated dogs, and to formulate recommendations for adjusting the vaccination schedule using mathematical modeling (MM). We used a fixed-effects modeling procedure to estimate the two-compartment model parameters using log-transformed data (obtained by RFFIT, rapid fluorescent focus inhibition test; and FAVN, fluorescent antibody virus-neutralization test) on the VNAs levels in the serum of vaccinated dogs. More vigorous immune response after a two-dose primary vaccination is formed in juvenile dogs at the age of 3 months to 1 year compared to the adult dogs. Following the primary vaccination and revaccination 1 year after, VNAs were produced more intensively in adult stray dogs than in domestic dogs. The short-term immune response observed in dogs aged up to 3 months is due to the presence of colostral antibodies and the active growth of the organism at this age. The results of our study confirm that most of the dogs have a level of antirabies VNAs of ≥0.5 IU/ml up to two or more years following immunization. However, only regular annual revaccination ensures the protective VNAs level in animals that responded poorly to vaccination due to various factors. The following antirabies vaccination schedule is recommended: primary vaccination of the dog at the age of 3 months up to 1 year with 1-2 month intervals, then revaccination annually. This work also demonstrates the possibility of a wider application of MM methods for solving problems of vaccine prevention.

Predicting the presence and titre of rabies virus-neutralizing antibodies from low-volume serum samples in low-containment facilities.

Serology is a core component of the surveillance and management of viral zoonoses. Virus neutralization tests are a gold standard serological diagnostic, but requirements for large volumes of serum and high biosafety containment can limit widespread use. Here, focusing on Rabies lyssavirus, a globally important zoonosis, we developed a pseudotype micro-neutralization rapid fluorescent focus inhibition test (pmRFFIT) that overcomes these limitations. Specifically, we adapted an existing micro-neutralization test to use a green fluorescent protein-tagged murine leukaemia virus pseudotype in lieu of pathogenic rabies virus, reducing the need for specialized reagents for antigen detection and enabling use in low-containment laboratories. We further used statistical models to generate rapid, quantitative predictions of the probability and titre of rabies virus-neutralizing antibodies from microscopic imaging of neutralization outcomes. Using 47 serum samples from domestic dogs with neutralizing antibody titres estimated using the fluorescent antibody virus neutralization test (FAVN), pmRFFIT showed moderate sensitivity (78.79%) and high specificity (84.62%). Despite small conflicts, titre predictions were correlated across tests repeated on different dates both for dog samples (r=0.93) and in a second data set of sera from wild common vampire bats (r=0.72, N=41), indicating repeatability. Our test uses a starting volume of 3.5 µl of serum, estimates titres from a single dilution of serum rather than requiring multiple dilutions and end point titration, and may be adapted to target neutralizing antibodies against alternative lyssavirus species. The pmRFFIT enables high-throughput detection of rabies virus-neutralizing antibodies in low-biocontainment settings and is suited to studies in wild or captive animals where large serum volumes cannot be obtained.

Adaptability of a field isolate of Classical swine fever virus to PK-15 cells

Classical swine fever (CSF) is an economically important pig disease affecting rural pig farming and vaccines are not freely available for control. In the present study an Indian isolate of CSF virus was passaged 75 times in PK-15 cells. At different passages, presence of virus was confirmed by Fluorescent Antibody Virus Neutralization (FAVN) test and Reverse Transcription-Polymerase Chain Reaction (RTPCR) for NS5B, E2 and 5-UTR genes. TCID50 titers were found to range between 4.00 and 8.97 at 10th and 75th passages respectively. Back passage and pathogenicity studies in susceptible pigs, the natural host, indicated that the virus was found to be attenuated following PK-15 passages and did not produce any clinical signs after 45th and 75th passages.

Detection of rabies antibodies in wild boars in north-east Romania by a rabies ELISA test

BackgroundIn the last few decades, Romania has been considered one of the European countries most affected by animal rabies, but a combination of oral rabies vaccination (ORV) campaigns in foxes alongside mandatory vaccination of pets has substantially decreased the number of rabies cases in recent years.The objective of this study was to detect rabies antibodies in wild boar serum and thoracic fluid samples collected during the hunting season after ORV campaigns in north-eastern Romania in order to identify if wild boars are substantial competitors to foxes for ORV baits.ResultsWhen the 312 wild boar samples were tested by ELISA (BioPro ELISA, Czech Republic), 42.31% (132/312) demonstrated rabies antibodies. In order to compare these wild boar results in terms of the percentage of immunisation, fox samples were also included in the study, and in this case only 28.40% (98/345) demonstrated rabies antibodies by ELISA. To check the diagnostic sensitivity and specificity of this ELISA, those samples with a sufficient volume from both species that had tested either negative or positive with an initial ELISA were then tested with the Fluorescent Antibody Virus Neutralisation (FAVN) assay. The overall concordance between the BioPro ELISA and FAVN test was 74.26% (75/101) in wild boar samples and 65.66% (65/99) in fox samples, 140 out of 200 samples being correlated with the two methods, although no significant statistical difference (p = 0.218) between the two species was registered. We found a good agreement by both tests for the ELISA-positive samples (91.30%), however the situation was different for the ELISA-negative samples, where a low agreement was demonstrated (41.18%).ConclusionsThis study reports for the first time the presence of rabies antibodies in wild boar samples collected during the hunting season in Romania after ORV campaigns in rabies endemic areas. It is also the first study to demonstrate that ELISA BioPro can be used on wild boar samples with satisfactory results compared to the FAVN test for this species.

Assessment of naturally acquired neutralizing antibodies against rabies Lyssavirus in a subset of Nunavik’s Inuit population considered most at risk of being exposed to rabid animals

Contact with infected saliva through the bite of a rabid animal is the main route of infection with the rabies Lyssavirus in humans. Although a few individuals have survived the infection, rabies remains the most lethal zoonotic infection worldwide. Over the last century, the dogma that rabies is invariably fatal has been challenged by the survival and recovery of infected animals. In humans, 11 studies have found rabies virus-specific antibodies in unvaccinated individuals exposed to rabies virus reservoir species, suggesting the possibility of asymptomatic rabies virus infection, contact with non-infectious virus or exposure to the virus without viral replication. Two of these studies were conducted in Arctic hunters. Considering the extensive exposure of Nunavik's Inuit to potentially infected animals through hunting, trapping, skinning and the preparation of Arctic carnivores, we analysed archived serum samples from the 2004 Nunavik Inuit Health Survey for the presence of rabies virus-neutralizing antibodies (rVNA) in this sub-population. A total of 196 participants who were considered at highest risk for exposure to rabies virus were targeted. Serum samples were tested for the presence of rVNA using a variation of the fluorescent antibody virus neutralization test, an assay recommended for the quantification of neutralizing antibody titres following vaccination. Our study identified two seropositive individuals among the 196 participants but a review of their medical record and a phone interview revealed previous vaccination. Our results do not provide evidence for naturally acquired rVNA in Nunavik's Inuit population.

Seroprevalence, cross antigenicity and circulation sphere of bat-borne hantaviruses revealed by serological and antigenic analyses.

Bats are newly identified reservoirs of hantaviruses (HVs) among which very divergent HVs have been discovered in recent years. However, their significance for public health remains unclear since their seroprevalence as well as antigenic relationship with human-infecting HVs have not been investigated. In the present study archived tissues of 1,419 bats of 22 species from 6 families collected in 5 south and southwest provinces in China were screened by pan-HV RT-PCR following viral metagenomic analysis. As a result nine HVs have been identified in two bat species in two provinces and phylogenetically classified into two species, Laibin virus (LAIV, ICTV approved species, 1 strain) and Xuan son virus (XSV, proposed species, 8 strains). Additionally, 709 serum samples of these bats were also analyzed by ELISA to investigate the seroprevalence and cross-reactivity between different HVs using expressed recombinant nucleocapsid proteins (rNPs) of LAIV, XSV and Seoul virus (SEOV). The cross-reactivity of some bat sera were further confirmed by western blot (WB) using three rNPs followed by fluorescent antibody virus neutralization test (FAVNT) against live SEOV. Results showed that the total HV seropositive rate of bat sera was 18.5% (131/709) with many cross reacting with two or all three rNPs and several able to neutralize SEOV. WB analysis using the three rNPs and their specific hyperimmune sera demonstrated cross-reactivity between XSV/SEOV and LAIV/XSV, but not LAIV/SEOV, indicating that XSV is antigenically closer to human-infecting HVs. In addition a study of the distribution of the viruses identified an area covering the region between Chinese Guangxi and North Vietnam, in which XSV and LAIV circulate within different bat colonies with a high seroprevalence. A circulation sphere of bat-borne HVs has therefore been proposed.

Efficacy of rabies vaccine with differing antigen payloads on pre and simulated post-exposure prophylaxis in cattle

Rabies is a fatal disease of humans and animals. Over the years various vaccines and vaccination procedures such as pre-and post-exposure prophylaxis (PrEP and PEP) have been used to control the losses incurred due to rabies. Unlike in humans, no firm PrEP and PEP guidelines are available for control of rabies in animals. In this study, healthy cattle calves were vaccinated with either single (PrEP regime) or five doses (simulated PEP regime) of rabies vaccine with a potency of 1.2 IU/dose or 2.7 IU/dose. Vaccine was administered on Day 0, 3, 7, 14 and 28 as per ‘Essen’ regimen. The serum was collected from all the vaccinated cattle on 0, 7, 14, 28 and 60 days post inoculation (dpi) and virus neutralisation antibody (VNA) levels were determined by fluorescent antibody virus neutralisation test (FAVN). The results reveal that vaccination with single dose of 2.7 IU/dose of rabies vaccine induced higher VNA in serum compared with 1.2 IU/dose vaccine. Comparison of serum antibody levels between animals vaccinated with single dose of high antigen payload (2.7 IU/dose) and multiple vaccination with 1.2 IU/dose vaccine showed significantly higher antibody levels in the former at 14, 28 and 60 dpi intervals. From this study, it is concluded that the protective levels of immunity in cattle can be achieved with high antigen payload vaccines with either two or three doses, respectively, in PrEP and simulated PEP programs.

Generation and Characterization of a Neutralizing Monoclonal Antibody Against Rabies Virus

Rabies is a zoonotic disease, for which effective treatment methods after the onset of clinical symptoms have not been developed yet. Polyclonal sera, both human and equine, along with vaccines are important means of disease prophylaxis. However, due to adverse reactions to the immunoglobulins of animal origin, high cost, and limited availability of the safer human serum, polyclonal antibodies should be substituted for a stable and efficient preparation, which is recombinant neutralizing antirabies monoclonal antibodies (mAbs). This paper reports generation of the humanized mAb 1C5, which binds with the antigenic site (AS) III of the rabies virus glycoprotein (RABVG) and demonstrates high virus neutralization activity in the fluorescent antibody virus neutralization test, as a result of expression in the Chinese hamster ovary (CHO) cells.

Development of a Blocking ELISA for Measuring Rabies Virus-specific Antibodies in Animals

Rabies virus (RABV)-specific antibodies in animals and humans are measured using standard methods such as fluorescent antibody virus neutralization (FAVN) tests and rapid fluorescent focus inhibition tests, which are based on cell culture systems. An alternative assay that is safe and easy to perform is required for rapid sero-surveillance following mass vaccination of animals. Two purified monoclonal antibodies (4G36 and B2H17) against RABV were selected as capture and detection antibodies, respectively. A genetically modified RABV, the ERAGS strain, was propagated and concentrated by polyethylene glycol precipitation. Optimal conditions for the RABV antigen, antibodies, and serum dilution for a blocking enzymelinked immune sorbent assay (B-ELISA) were established. We evaluated the sensitivity, specificity, and accuracy of the B-ELISA using serum samples from 138 dogs, 71 raccoon dogs, and 25 cats. The B-ELISA showed a diagnostic sensitivity of 95.8-96.3%, specificity of 91.3-100%, and accuracy of 96.0-97.2% compared to the FAVN test. These results suggest that the B-ELISA is useful for sero-surveillance of RABV in dogs, raccoon dogs, and cats.

A genetically modified rabies vaccine (ERAGS) induces protective immunity in dogs and cattle.

PurposeThe current live attenuated rabies vaccine must be replaced with a safer vaccine based on the ERAGS strain to prevent rabies in South Korea. We evaluated the safety and immunogenicity of a new strain in dogs and cattle.Materials and MethodsThe ERAGS strain, featuring two mutations altering two amino acids in a glycoprotein of rabies virus, was propagated in NG108-15 cells. We lyophilized the virus in the presence of two different stabilizers to evaluate the utilities of such preparations as novel rabies vaccines for animals. To explore safety and immunogenicity, dogs and cattle were inoculated with the vaccine at various doses via different routes and observed daily for 8 weeks post-inoculation (WPI). Immunogenicity was evaluated using a fluorescent antibody virus neutralization test or enzyme-linked immunosorbent assay.ResultsThe two different stabilizers did not differ greatly in terms of maintenance of virus viability in accelerated stability testing. No clinical signs of rabies developed in dogs or cattle inoculated with the vaccines (107.0 FAID50/mL). Dogs and cattle inoculated intramuscularly with 105.0 FAID50/mL exhibited virus neutralization assay titers of 4.6 IU/mL and 1.5 to 0.87 IU/mL at 4 WPI, respectively. All control animals remained rabies virus–seronegative throughout, confirming that no contact transmission occurred between vaccinated and control animals.ConclusionOur findings indicate that the new rabies vaccine is safe and immunogenic in dogs and cattle.

Natural exposure of bats in Grenada to rabies virus

ABSTRACT Introduction: Grenada is a rabies endemic country, where terrestrial rabies is maintained in the small Indian mongoose (Herpestes auropunctatus). The role of bats in the epidemiology of rabies in Grenada is unknown. A 1974 report described one rabies virus positive Jamaican fruit bat (Artibeus jamaicensis), and a high seroprevalence in this species. In the current study, the natural exposure to rabies virus in Grenadian bats was re-evaluated. It is postulated that bats serve as a natural rabies reservoir, probably circulating a bat-specific rabies virus variant. Material and methods: Bats were trapped in 2015 in all six parishes of Grenada using mist- and hand nets. For the detection of rabies virus in brain tissue, the direct fluorescent antibody test (dFAT) and the reverse transcription polymerase chain reaction (RT-PCR) were used. Serum neutralizing antibodies were determined using the fluorescent antibody virus neutralization test (FAVN). Results and discussion: Brain tissue and sera from 111 insectivorous and frugivorous bats belonging to four species were tested (52 Artibeus jamaicensis, two Artibeus lituratus, 33 Glossophaga longirostris, 24 Molossus molossus). Rabies virus antigen and genomic RNA were not detected in brain tissues. Rabies virus neutralizing antibodies were detected in the sera of eight A. jamaicensis in four of the six parishes. Bats in Grenada continue to show natural exposure to rabies virus. As rabies virus was not isolated in this study, serology alone is not sufficient to determine the strain of rabies virus circulating in A. jamaicensis bats in Grenada. Conclusion: Artibeus jamaicensis appears to play a role as a reservoir bat species, which is of public health concern in Grenada. Dispersion of bats to neighboring islands is possible and serological bat surveys should be initiated in these neighboring states, especially in those areas that are free of rabies in terrestrial mammals.

A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples.

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.

Factors associated with dog rabies immunisation status in Bamako, Mali

We conducted a cross-sectional survey in Bamako, Mali, to determine for the first time the seroprevalence of rabies virus antibodies in the dog population and people's knowledge, attitudes and practices (KAP) towards the disease and its control. Antibody detection was done with the fluorescent antibody virus neutralisation (FAVN) test, with a positivity threshold of 0.25IU/ml.We visited 2956 households in 2010 and 2011 and found 379 dogs in 279 households. Data were collected on 279 dog-owning households, on 1017 non-dog-owning households and on 311 dogs. A serum or plasma sample was collected from 98 dogs. For 26 dogs we had sufficient data to describe the antibody decline over time after rabies vaccination using a quadratic regression.Ninety percent of interviewed persons (95% CI: 85%–91%) knew about rabies. The majority of interviewees knew that rabies is transmitted from dogs to humans, and some of the characteristic clinical signs seen in rabid dogs (change of behaviour, biting, salivation) could be listed by the majority. When asked how people behave regarding a rabid dog, killing the animal was the most frequent answer (>70%). Most (65% of the non-dog-owners and 81% of the dog-owners) were aware that vaccination of dogs can prevent rabies, but only a minority of dog-owners could answer correctly at what age the dog should get a first rabies vaccination (i.e. at 3 months). There was also strong consensus among dog-owners that it is better to protect their dog from becoming rabid by vaccinating it rather than needing to treat a bitten person. Forty-five percent (n=306; 95% CI 38%–52%) of dogs were reported as vaccinated against rabies at least once, but less than half of these (59/136) had a valid vaccination card. When asked for reasons for non-vaccination, cost was the most frequent reason at 31% (95% CI: 21%–43%), while general negligence was mentioned by 15% (95% CI: 10%–24%). Approximately one third of dog-owners would not pay for vaccination. To reach a threshold of 70% of vaccinated owned dogs, vaccination should not cost more than 0.2€ (100 FCFA).The seroprevalence of rabies virus antibodies in the examined dog population was low: 24% (n=98; 95% CI 15%–36%) with titres ≥0.25IU/ml and was 46% (n=39; 95% CI 29%–63%) when only including those reported as vaccinated by their owners. A seroprevalence of 59% (n=18; 95% CI 33%–80%) was reached if the analysis included only dogs with a valid vaccination certificate. Interestingly 4/22 dogs showed titres ≥0.25IU/ml despite being reported as unvaccinated. The Rabisin® vaccine showed generally higher IU titres than the Dog Vac Rabia® vaccine. All animals after booster vaccination had titres ≥0.25IU/ml which was not the case in primo-vaccinated animals. For the Rabisin® vaccine, a Kaplan Meier estimate suggested that to maintain an antibody titre of ≥0.25IU/ml for 75% of owned dogs, revaccination should be done after not more than 2.5 years.This work contributes vital information towards planning an effective dog rabies control programme for the district of Bamako.

Antibody Response of Dogs to Ethiorab Rabies Vaccine

Rabies is 100% fatal, but it is preventable. More than 95% of human rabies cases occur in improperly treated individuals. This is partly due to the fact that modern post-exposure rabies prophylaxis is expensive and therefore not readily available in many endemic regions. Nervous tissue vaccine has been in use for more than 100 yrs. These vaccines have now been superseded in purity, potency, immunogenicity and safety. The efficacy and immunogenicity of inactivated tissue culture vaccine, produced in Ethiopia was evaluated. Twelve experimental dogs from local breed were duly conditioned during a quarantine period and assigned to two groups randomly. Animals in group I (cases) were vaccinated subcutaneously with 1 ml of our experimental vaccine. Dogs in group II served as non-vaccinated controls. The immune response of each dog was monitored for 120 days. On the day 120 after final sampling, all dogs were challenged in the masseter muscle with a rabies street virus of canine origin. To evaluate the titer of the rabies virus neutralizing antibodies (VNA), sera were analyzed by Fluorescent Antibody Virus Neutralization (FAVN) Test. Geometric Mean Titers (GMT) to rabies virus was determined at days 7, 15, 21, 30, 60, 90 and 120. Geometric mean titers were equal to 1.59, 1.73, 2.19, 3.58, 3.17, 3.35 and 3.56 IU/ml respectively. All dogs showed VNA titers higher than the 0.5 IU/ml mandated WHO recommended threshold. 83.3% vaccinated dogs, survived the challenge virus. In contrast, all dogs in the control (non-vaccinated group), developed rabies and died. This study indicated cell culture-based anti-rabies vaccine manufactured in Ethiopia is efficacious and immunogenic.

Detection of rabies antibodies in dog sera.

In the presented work, we compared the results of determination of rabies antibodies using three in vitro methods: rapid fluorescent focus inhibition test (RFFIT), fluorescent antibody virus neutralisation test (FAVNT) and the immunoenzymatic assay (ELISA). 196 dog sera samples were examined with FAVNT, RFFIT methods and the ELISA test. Sera with low and sufficiently high titre of antibodies had a similar result in determining by all methods. A critical level of rabies antibodies close to the required protection level (0.5 IU/cm3) was seen in sera of 18 dogs (9.18%); these were the sera obtained after primary vaccination of dogs. At this level, even small differences can cause a change in the assessment of the patient's serum seronegativity or seropositivity. Therefore, it is important to choose the appropriate method that has sufficiently strict criteria while having a good reproducibility.