Detection Of Rabies Antibodies Research Articles

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.

First international collaborative study to evaluate rabies antibody detection method for use in monitoring the effectiveness of oral vaccination programmes in fox and raccoon dog in Europe

The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97.2% and 97.5%, respectively. The overall agreement values obtained for the four marketed oral vaccines used in Europe were all equal to or greater than 95%. The coefficients of concordance obtained by laboratories ranged from 87.2% to 100%. The results of this collaborative study show good robustness and reproducibility of the BioPro ELISA Rabies Ab kit.

Estimation of Rabies Antibodies in Animal Sera Using Different Techniques

Rabies antibodies were detected in dog, cat and cattle serum samples and quantified through application of four different methods including Serum neutralization test (SNT), rapid fluorescence focus inhibition test (RFFIT), indirect ELISA and Dot ELISA. The comparison of rabies antibody titers determined in tested samples using RFFIT showed lower than indirect ELISA and SNT. The dot ELISA may have potential applications as a rapid, simple and economical field test in the detection of rabies antibodies but it revealed lower sensitivity and specificity than indirect ELISA and SNT but more than RFFIT. Otherwise, RFFIT needs more time and expensive test. So, the previous study confirmed that indirect ELISA is the best of choice for detection of rabies antibodies in sera of different animal species because of its highly sensitive, specific and safe laboratory technique.

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.

Use of filter paper blood samples for rabies antibody detection in foxes and raccoon dogs

The effectiveness of oral rabies vaccination in wildlife is usually evaluated by the detection of rabies antibodies. However, the assessment of rabies antibodies has several technical difficulties in the field, such as the collection, storage, transport and titration of blood samples, often of poor quality.The objective of this study was to assess the feasibility of collecting blood on a filter paper (FP) coupled with enzyme-linked immunosorbent assay (ELISA) titration of rabies antibodies in raccoon dogs and red foxes.The FP blood sampling method was found highly specific and repeatable in both species. Overall, results obtained with the FP sampling method were highly concordant with the conventional (venipuncture) sampling methods. Blood eluates from FP samples from foxes and raccoon dogs tested using ELISA showed concordance values of 92% and 95%, respectively, with serum samples tested using the seroneutralisation test and values of 95% and 91%, respectively, when the ELISA was used on both types of sample.The use of FP blood sampling coupled with the titration of rabies antibodies by ELISA provides a reliable alternative to conventional blood sampling and serum testing by seroneutralisation. This simple procedure is particularly attractive and cost-effective for assessing the effectiveness of oral rabies vaccination in field conditions.

Rural Rabies Prevention Project - A ‘One Health’ Experiment in India: An Overview

Aim: To assess the feasibility of implementing approach to prevent human rabies and control animal rabies in a rural community. Study Design: Health research in a rural setting. Place and Duration of Study: A medical college and a veterinary college along with an animal welfare organization delivered a wide array of integrated services in three villages' i.e. Kumbalagodu, Thagachikuppe and Gerupalya near Bangalore, India comprising a population of 10,220 persons for a period of two years from December, 2009 to November, 2011. The nearby three villages of Ramohally, Vinayakanagara and Bhimanakuppe with a population of 6,023 persons formed the control group, with no project inputs. Methodology: This consisted of household surveys at the beginning and end of two years ; rabies awareness campaigns; clinical and laboratory surveillance of rabies in dogs; rabies post-exposure prophylaxis (PEP) in humans; pre-exposure rabies prophylaxis (PrEP) by intradermal route in school children, pet dog owners and veterinarians ; mass dog vaccination and deworming; and sero-surveillance in both dogs and humans. Results: The ratio of veterinary and medical manpower in study villages was 1: 11 and dog to human ratio was 1: 23.The information, education and communication materials developed and used were domestic outdoor wall writings (11), domestic indoor annual wall calendar (2000), school book labels (1000); game charts (16); wall posters (65); flip chart (15); rabies educational DVD (1). There were 102 local cable television transmissions on rabies prevention. 69 persons received rabies PEP. PrEP was given to 368 school children, pet dog owners and veterinarians. Sixty one human serum samples were analyzed by rapid fluorescent focus inhibition test for rabies antibody detection. Sixteen veterinarians were trained to use direct rapid immunohistochemical test (dRIT) for rabies diagnosis. Six ruminants were confirmed rabid by dRIT. There were no cases of human rabies. The cost of entire project was US $ 85,958. Conclusion: A blend of medical, veterinary and animal welfare were successfully delivered through a health approach. Based on this success a conceptual model was evolved to propagate its replication in other rural communities across India.

Evaluation of ELISA for detection of rabies antibodies in domestic carnivores

Serological tests of pets have increased as many rabies-free countries have amended their quarantine measures and adopted a scheme requiring rabies vaccination followed by a serological test. A European directive requires the measurement of neutralising antibodies as proof of protection to allow the free movement of pets within the European Union and between third countries non listed in the list C of regulation 998/2003 and European countries. At present, the recommended neutralisation tests (FAVN test or RFFIT) are time-consuming, expensive and require highly trained technicians as well as special laboratory facilities. The rabies ELISA designed by BioPro was developed initially for use for field samples from foxes to check the efficacy of oral vaccination campaigns in Europe. In this study, the specificity, sensitivity and reliability of this commercial rabies ELISA was evaluated for testing sera from dogs and cats involved in international trade. The specificity evaluated in 315 unvaccinated animals was 100%. Concordance of 86.2% was obtained when comparing BioPro ELISA to the gold standard FAVN test in 701 samples from vaccinated dogs and cats. The rabies ELISA developed recently can be considered a valuable method for the assessment of rabies antibodies in vaccinated domestic carnivores in combination with neutralisation tests.

Immunoperoxidase inhibition assay for rabies antibody detection

An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG–peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the “simplified fluorescence inhibition microtest” (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available.

A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa

The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r=0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.

An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies

The envelope glycoprotein G of rabies virus in vaccines induces the production of neutralizing antibodies important in the protection against the disease. The measurement of anti-envelope glycoprotein antibodies is a good predictor of the degree of humoral immunity in people during anti-rabies treatment or after vaccination. Several assays exist for the serological determination of antibody protection against rabies virus infection. Antibody neutralization by the rapid fluorescent focus inhibition test (RFFIT) or the fluorescent antibody virus neutralization (FAVN) test is currently the gold standard. Performance of the highly complex RFFIT and FAVN tests, however, requires specialized reference laboratories with expertise with this assay. Although not widely used, ELISA test kits are available and may be an additional option for testing that is more accessible. The aim of the present study was to evaluate available ELISA assays for the determination of anti-rabies antibodies. We compared the Bio-Rad Platelia Rabies II ELISA, DRG Rabies Virus IgG Ab ELISA and Focus Diagnostics Rabies Antibody Detection by ELISA to RFFIT. Bland-Altman plots comparing the Bio-Rad Platelia assay and the Focus Diagnostics assay to RFFIT showed a low degree of variability between the ELISA assays and RFFIT results except in samples with high RFFIT values. The agreement, sensitivity and specificity of Bio-Rad Platelia Rabies II ELISA when compared to RFFIT were 95.1 %, 94.1 % and 95.8 %, respectively. The DRG Rabies assay compared to RFFIT had an agreement of 77.7 %, a sensitivity of 86.7 % and a specificity of 69.4 %. The agreement, sensitivity and specificity of Focus Diagnostics Rabies Detection by ELISA when compared to RFFIT were 82.2 %, 91.7 % and 73.0 %, respectively. Overall, the Bio-Rad Platelia assay showed higher accuracy and specificity than either the DRG or Focus assays. All of these ELISAs, however, measure all antibody types and do not discriminate the neutralizing antibodies as measured by functional assays (RFFIT and FAVN) and cannot be relied upon to predict the neutralizing activity of the sera. The results of this study offer insight into the availability of alternative, less-complex methods to monitor rabies antibody titres in at-risk individuals following vaccination.

Serological survey for rabies in serum samples from vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

The chiropterans constitute 25% of the world's mammal fauna. Due to the destruction of their natural ecosystem, the vampire bats have moved from nature to artificial roosts closer to man and domestic animals. This phenomenon has happened particularly in rural areas. Rabies is a viral anthropozoonosis, 100% lethal, and vampire bats (Desmodus rotundus) represent an important role in its epidemiology. D. rotundus were captured at night with mesh nets in partnership with the Botucatu Defense Office and sent to the Zoonosis Diagnostic Service, at the School of Veterinary Medicine and Animal Husbandry, UNESP. Serum samples from 204 bats were analyzed by enzyme-linked immunosorbent assay (ELISA) and fluorescent antibody viral neutralization test (FAVN) for rabies antibody detection. The results showed 7.4% of sera with titers higher or equal to 0.5 U for rabies antibodies, which demonstrated viral flow circulation among the studied region. Data suggest a need for constant monitoring accomplished by epidemiological and sanitary measures.

Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats

A protocol suitable for the detection of rabies virus-specific antibodies in serum samples from companion animals using an enzyme linked immunosorbent assay (ELISA) is described. This method has been used successfully for the qualitative assessment of rabies virus-specific antibodies in serum samples from a cohort of vaccinated dogs and cats. In two initial field studies, a variable population of field samples from the Veterinary Laboratories Agency (VLA), United Kingdom were tested. In the first study ( n=1000), the number of false-positive and false-negative results was 11 samples (1.1%) and 67 samples (6.7%), respectively. In the second study ( n=920), the number of false-positive and false-negative results was 7 samples (0.8%) and 52 samples (5.7%). In a third study, undertaken at l’Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Nancy, France ( n=440), 1 false-positive sample (0.23%) and 91 (20.7%) false-negative samples were identified. Data generated using this prototype ELISA indicate a strong correlation for specificity when compared to the gold standard fluorescent antibody virus neutralisation (FAVN) test. Although the ELISA has a lower sensitivity than the FAVN test, it is a useful tool for rapidly screening serum samples from vaccinated companion animals. Using a cut-off value of 0.6 EU/ml, the sensitivity ( R=93% from VLA and 79% from AFSSA) and specificity ( R=97.3%) indices between the ELISA compared favourably with data generated using the FAVN test. The major advantages of the ELISA test are that it is a qualitative tool that can be completed in four hours, does not require the use of live virus and can be performed without the need for specialised laboratory containment. This contrasts with 4 days using conventional rabies antibody virus neutralisation assays. Using the current format, the ELISA assay described would be a valuable screening tool for the detection of rabies antibodies from vaccinated domestic animals in combination with other Office International des Epizooties (OIE) accepted serological tests.

Dog ecology and demography information to support the planning of rabies control in Machakos District, Kenya

A study of 150 dog-owning households from six randomly selected sublocations was conducted in Machakos District, Kenya. Initially, all households were visited to collect information on dog ecology and demography based on WHO guidelines and to collect serum for rabies antibody detection. A second visit was made 1 year later, to obtain follow-up data on births, deaths, dog movements and other events since the first visit. Dog ownership was common, with a range of 53–81% (mean=63%) of households owning dogs in the six sublocations. Dog density for the five more rural sublocations ranged from 6 to 21 dogs km −2 and for the peri-urban sublocation was 110 dogs km −2. The dog population was estimated to be growing at 9% p.a. (95% C.I. 4–14%). This growth was a function of very high fecundity (1.3 females per female per year) more than compensating for high mortality, particularly among females. Life expectancy from birth was 3.5 years for males and 2.4 years for females. Half the dogs at any one time were less than 1 year of age. All dogs, by design of the study, were owned. Of these, 69% were never restricted and roamed freely to forage for food and mix with other dogs. Only a small proportion of dogs (5%) were fed commercial dog food. Most households reported observing dogs scavenging their garbage, including: their own dogs (81%), their neighbours’ dogs (75%) and unknown dogs (45%). Only 29% of dogs at least 3 months of age were reported to be vaccinated against rabies. The proportion vaccinated varied widely between sublocations (5–68%); 48% of dogs reportedly vaccinated had detectable antibodies, 31% at or above levels considered to indicate seroconversion. The proportion of dogs with detectable antibodies declined according to the time since last vaccination (55% if vaccinated ≤1 year, 47% ≤2 years and 36% >2 years); 20% of dogs reported not to have been vaccinated had detectable rabies antibody. Compared to other dog populations in rural eastern and southern Africa, Machakos District has a high density of dogs. The Machakos dog population is growing, highly dynamic, poorly supervised and inadequately vaccinated against rabies. The main implication for rabies control is that adequate vaccination coverage is unlikely to be achieved, even under optimal delivery, using the current strategy of annual vaccination of dogs older than 3 months.

Using the UV ParaLens ® adapter on a standard laboratory microscope for fluorescent rabies antibody detection

Using the UV ParaLens ® adapter on a standard laboratory microscope for fluorescent rabies antibody detection

A rabies agglutination test (RAT) for rabies antibody detection

An agglutination test has been developed for the detection of rabies antibodies aftet human vaccination. The rabies agglutination test (RAT) is based on the capability of specific antibody to agglutinate sensitized polystyrene (or latex) beads. In the RAT, latex beads were coated, in a first step, with inactivated and purified rabies virus (PV strain adapted and propagated on BHK-21 cells) and, in a second step, with bovine serum albumin. Negative control beads were coated with bovine serum albumin (BSA) only. To test for human antibody, several microliters of serum were mixed on a glass slide with an equal volume of virus-sensitized beads and the mixture was gently agitated. After a few minutes, agglutination was visible with sera which had been characterized as positive by the virus neutralization antibody (VNAb) technique. No agglutination was observed with negative sera tested with virus-coated beads or with positive sera tested with BSA-coated beads. Virus-sensitized beads were agglutinated when the virus neutralizing antibody titres were equal to or greater than 2·5 international units per ml (IU/ml) in human sera. The concordance between the RAT results and VNAb titres was about 97% when 2·5 IU/ml was taken as the cut off value for determining the positive sera with the VNAb technique. The possibility that clinicians might use the RAT as a simple means to determine sero-conversion at the end of the post-exposure treatment of patients is discussed.

Rapid test for detection of rabies antibodies in human serum.

A simple, sensitive, rapid method based on the principle of immunoadherence hemagglutination (IAHA) has been devised for the detection of rabies antibody. In this test, fixation of complement to complexes of rabies antigen with specific antibodies is readily detected by agglutination of human erythrocytes bearing receptors for C3. Sera from individuals undergoing preexposure rabies immunization were tested for rabies antibodies by the IAHA method and by a virus neutralization test performed in tissue culture, the rapid fluorescent focus inhibition test. IAHA titers showed a high degree of correlation with rapid fluorescent focus inhibition test titers, although it is not known whether results of the IAHA test represent the detection of neutralizing antibodies. An advantage of the IAHA test over the rapid fluorescent focus inhibition test was that results were obtained in a shorter period of time. In some instances, this can be of clinical significance in determining antibody levels to rabies virus. Furthermore, the IAHA test is most applicable as a rapid screening tool for the detection and quantitation of rabies antibodies in vaccinated subjects.

Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM results

Techniques usually employed for the detection of rabies' antibodies are costly, time consuming, and sometimes fail to detect early antibodies. The introduction of immunoenzymatic techniques in the serology of viral disease represents a new and important advance. We therefore adapted this technique to the detection of rabies antibodies. We have found that the sera from rabies patients who had not received antirabies treatment do not show seroneutralizing antibodies until several days after the onset of symptoms. However, antibodies can be detected some days earlier by the immunoenzymatic method in the same samples. Furthermore, the immunoenzymatic test was applied to the detection of both the IgM or the IgG class of antirabies antibodies using an antihuman Ig-or antihuman IgG-peroxydase conjugate.