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This pioneering study is prompted by the imperative to enhance our understanding of a constituent within Ukraine's rabies control strategy, specifically focusing on the vaccination of free-roaming canines against rabies with a local vaccine in certain endemic rabies areas. The cross-sectional study encompassed the capture, sterilization, deworming, and rabies vaccination processes in 160 free-roaming dogs, followed by the collection of blood samples to examine RABV-specific neutralising antibodies in 16 dogs (10% of the vaccinated population), captured from sectors (territories) with a higher density of population and with higher density of previously vaccinated free-roaming dogs. Half of the targeted samples comprised males (n = 8), while the remaining half consisted of females (n = 8). The median of virus-neutralising antibody level was 0.58 IU/mL, with a minimum protective threshold of 0.5 IU/mL. Antibody titers below the 0.5 IU/mL threshold were detected in 25.0% (2/8) of the male group, and in 62.5% (5/8) of the female group. Notably, male dogs exhibited a higher median antibody level of 0.66 IU/mL, compared to females, who presented a median level of 0.26 IU/mL. However, no statistically significant difference was found between the male and female groups (P = 0.36). In general, more than half of the tested population (56.3%) exceeded the 0.5 IU/mL protective threshold 4 months post-vaccination. The inadequate levels of antibodies neutralizing the rabies virus are likely a result of a confluence of factors, including stressors such as nutritional and temperature-related challenges, along with variations in the responses of individual immune systems. Considering the endemic rabies situation and large population of free-roaming dogs in Ukraine, we suggest repeated vaccination for free-roaming dogs against rabies one year after the previous vaccination. In perspective, we suggest conducting large-scale epidemiological studies to assess the impact of animal-related, vaccine-related, and environment-related parameters on the efficacy of rabies vaccines used in Ukraine.

Background: Rabies, a potentially lethal virus, affects more than 150 countries. Although the rabies vaccine and immunoglobulin have been available since 1908, Bangladesh is new to vaccine manufacturing. We checked the quality of the local manufacturing rabies vaccine for substandard. Methods: The potency and immunogenicity of 20 vaccines were analyzed by three in vivo and in vitro methods from March 2020 to May 2023. Single radial immunodiffusion, fluorescent antibody virus neutralization, and national institutes of health tests were carried out to evaluate the vaccine's efficacy to provide sufficient protection against the rabies virus. Results: The potency of the rabies vaccine was determined by the in vitro SRID method by measuring glycoprotein content. An average of 16 articles from each batch was calculated. The minimum and maximum average mean values of the 20 batches were 5.058 and 5.346, respectively. The variance was calculated at 0.00566. We found a coefficient of variation (CV) between 9.36% and 14.80%. The 100% sample was satisfactory, as these samples had a potency of over 2.5 IU/mL. To observe immunogenicity, we applied the FAVN method for determining antibody titers. An average of 16 articles from every batch were counted to quantify antibody titers. The mean quantity of antibody titers ranged from 2.389 to 3.3875. The CV was slightly lower because of the dispersion of the data. At last, we performed an in vivo method, the NIH test method, to determine potency based on mortality rate. We found a mean value of 4.777 IU/SHD with a standard deviation of 1.13 IU/SHD. All 20 batches were found 100% satisfactory in the NIH test. Conclusion: The study implies that the rabies human vaccines manufactured in Bangladesh are potent enough to provide sufficient immunogenicity. Our research is warranted testimony for healthcare providers who work to extirpate rabies. [ABSTRACT FROM AUTHOR]

This pioneering study is prompted by the imperative to enhance our understanding of a constituent within Ukraine's rabies control strategy, specifically focusing on the vaccination of free-roaming canines against rabies with a local vaccine in certain endemic rabies areas. The cross-sectional study encompassed the capture, sterilization, deworming, and rabies vaccination processes in 160 free-roaming dogs, followed by the collection of blood samples to examine RABV-specific neutralising antibodies in 16 dogs (10% of the vaccinated population), captured from sectors (territories) with a higher density of population and with higher density of previously vaccinated free-roaming dogs. Half of the targeted samples comprised males (n = 8), while the remaining half consisted of females (n = 8). The median of virus-neutralising antibody level was 0.58 IU/mL, with a minimum protective threshold of 0.5 IU/mL. Antibody titers below the 0.5 IU/mL threshold were detected in 25.0% (2/8) of the male group, and in 62.5% (5/8) of the female group. Notably, male dogs exhibited a higher median antibody level of 0.66 IU/mL, compared to females, who presented a median level of 0.26 IU/mL. However, no statistically significant difference was found between the male and female groups (P = 0.36). In general, more than half of the tested population (56.3%) exceeded the 0.5 IU/mL protective threshold 4 months post-vaccination. The inadequate levels of antibodies neutralizing the rabies virus are likely a result of a confluence of factors, including stressors such as nutritional and temperature-related challenges, along with variations in the responses of individual immune systems. Considering the endemic rabies situation and large population of free-roaming dogs in Ukraine, we suggest repeated vaccination for free-roaming dogs against rabies one year after the previous vaccination. In perspective, we suggest conducting large-scale epidemiological studies to assess the impact of animal-related, vaccine-related, and environment- related parameters on the efficacy of rabies vaccines used in Ukraine. [ABSTRACT FROM AUTHOR]

Abstract Background Rabies (RABV) is an enzootic disease in Tunisia, with dogs being the primary reservoir. Vaccinating dogs is the key to eradicate rabies. Regional Veterinary Services conduct nationwide immunisation campaigns on an annual basis. Evaluation of the immune response is still important to make sure that the vaccination is effective in the conditions of the Tunisian field. In this paper, the FAVN technique was used to test rabies antibody dynamics in dogs from three distinct Tunisian areas observed for one year following a mass vaccination campaign. Results On day 30 after vaccination, 75% of all dogs vaccinated during the campaign were sero-positive (titres greater than or equal to 0.5 transformed IU/ml). On day 180, 48% of all dogs were sero-positive. Only 25.6% of primary-vaccinated dogs remained sero-positive on day 180 and 7% on day 365, whereas 91% of previously sero-positive dogs remained sero-positive on day 365. Conclusions Although a single rabies vaccine is successful at stimulating an immunological response, it is recommended that primary-vaccinated dogs have a second booster between one and three months after the initial vaccination to maintain seropositivity. To achieve the rabies eradication objective, all dogs should receive an annual booster to maintain effective immunological protection.

Background: Rabies (RABV) is an enzootic disease in Tunisia, with dogs being the primary reservoir. Vaccinating dogs is the key to eradicate rabies. Regional Veterinary Services conduct nationwide immunisation campaigns on an annual basis. Evaluation of the immune response is still important to make sure that the vaccination is effective in the conditions of the Tunisian field. In this paper, the FAVN technique was used to test rabies antibody dynamics in dogs from three distinct Tunisian areas observed for one year following a mass vaccination campaign. Results: On day 30 after vaccination, 75% of all dogs vaccinated during the campaign were sero-positive (titres greater than or equal to 0.5 transformed IU/ml). On day 180, 48% of all dogs were sero-positive. Only 25.6% of primary-vaccinated dogs remained sero-positive on day 180 and 7% on day 365, whereas 91% of previously sero-positive dogs remained sero-positive on day 365. Conclusions: Although a single rabies vaccine is successful at stimulating an immunological response, it is recommended that primary-vaccinated dogs have a second booster between one and three months after the initial vaccination to maintain seropositivity. To achieve the rabies eradication objective, all dogs should receive an annual booster to maintain effective immunological protection. [ABSTRACT FROM AUTHOR]

Limited data exist on the factors affecting feline rabies vaccination outcomes during primary immunization. This study aimed to assess if specific factors (signalment, vaccination count, vaccine brand, and time since last vaccination) correlated with meeting global antibody titer standards and absolute titers in young cats given monovalent inactivated rabies vaccines. Analyzing a dataset from cats tested before their first annual booster using the FAVN test, logistic and linear regression models were applied. Among 379 cats, 94.2 % achieved titers meeting or exceeding the standard threshold (≥0.5 IU/ml). Time since last vaccination proved to be the primary predictor of vaccination success. Cats receiving two vaccinations tended toward higher titers. Age, sex, breed, and vaccine type showed no impact on outcomes. The present study indicates that vaccination failure in young cats is uncommon, and that the time interval from the latest vaccination is the single most important predictor of successful rabies vaccination. • Rabies vaccination failures are uncommon in young cats. • Time from last vaccination to blood sampling predicts rabies vaccine outcome. • Cats vaccinated twice tended to have higher antibody titers. • Prior vaccinations number did not affect odds of attaining titer threshold. [ABSTRACT FROM AUTHOR]

Rabies is an almost invariably fatal disease. According to the World Health Organization (WHO), rabies virus neutralizing antibody (RVNA) titers of ≥0.5 IU/mL are considered adequate for rabies protection. Therefore, detection and quantification of RABV antibodies are important. Many methods have been developed for detecting RABV antibodies. In the present study, we reviewed several methods of detecting RABV antibodies in human and animal samples and evaluated and compared their performance. Of 34 methods, 5 demonstrated unsatisfactory sensitivity or specificity. The others exhibited sensitivity and specificity of ≥75%. The correlation coefficient for five of eight methods was >0.8. The Bland–Altman mean bias of five of five methods was <±2.0. The kappa values of 25 of 28 methods were higher than 0.4, demonstrating at least moderate agreement. Analysis of the performance of these methods emphasized that any new technology should be considered carefully and objectively before being used as an appropriate and applicable alternative. [ABSTRACT FROM AUTHOR]

Background: Fluorescent antibody virus neutralization (FAVN) test is a standard assay for quantifying rabies virus-neutralizing antibody (VNA) in serum. However, a safer rabies virus (RABV) should be used in the FAVN assay. There is a need for a new method that is economical and time-saving by eliminating the immunostaining step. Objectives: to improve the traditional FAVN method by rescuing and characterizing a new recombinant RABV expressing green fluorescent protein (GFP). Methods: A new recombinant RABV expressing GFP designated as ERAGS-GFP was rescued using a reverse genetic system. Immuno-fluorescence assay, peroxidase-linked assay, electron microscopy and reverse transcription polymerase chain reaction were performed to confirm the recombinant ERAGS-GFP virus as a RABV expressing the GFP gene. The safety of ERAGS-GFP was evaluated in 4-week-old mice. The rabies VNA titers were measured and compared with conventional FAVN and FAVN-GFP tests using VERO cells. Results: The virus propagated in VERO cells was confirmed as RABV expressing GFP. The ERAGS-GFP showed the highest titer (108.0 TCID50/mL) in VERO cells at 5 days postinoculation, and GFP expression persisted until passage 30. The body weight of 4-week-old mice inoculated intracranially with ERAGS-GFP continued to increase and the survival rate was 100%. In 62 dog sera, the FAVN-GFP result was significantly correlated with that of conventional FAVN (r = 0.95). Conclusions: We constructed ERAGS-GFP, which could replace the challenge virus standard-11 strain used in FAVN test. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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 n eutralisation t est using m C herry producing v irus (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. • A virus producing a fluorescent protein has been developed to measure neutralizing antibodies against rabies virus. • Using this simplified method, viral antigens can be detected without a labelled antibody allowing faster turnaround times and lower costs. • The results achieved were equivalent to a WOAH accepted fluorescent antibody virus neutralisation test (FAVN) • The serological assay can be automated using a cell imaging multi-mode reader. [ABSTRACT FROM AUTHOR]

The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

There is currently limited information on the factors influencing the outcome of rabies vaccination in dogs based on the primary immunization schedule. The objective of this study was to investigate whether selected variables (signalment, number of vaccinations, vaccine brand and multivalence, and time interval between the most recent vaccination and blood sampling) were associated with the achievement of an acceptable titer threshold (based on international standards) and with absolute antibody titers in young dogs vaccinated with commercially available vaccines. Serologic data from 662 dogs tested prior to their first annual booster for rabies were retrospectively reviewed. Neutralizing antibody titers were determined using a fluorescent antibody neutralization test. An acceptable titer threshold (≥0.5 IU/ml) was achieved in 86.5% of the dogs. Dogs that had been vaccinated twice had significantly ( P < 0.001) higher antibody titers compared with dogs vaccinated once. The odds of achieving seropositivity and the median absolute antibody titer tended to decrease with increasing time between vaccination and blood sampling. Dogs vaccinated with monovalent vaccines were more likely to achieve an acceptable titer than dogs vaccinated with polyvalent vaccines. Dogs that were vaccinated after 3–6 months of age were more likely to develop higher antibody titers. These results indicate that the administration of two vaccines rather than one vaccine in the primary immunization schedule for rabies, result in a superior vaccination response and may be a more beneficial policy for ensuring pre-exposure prophylaxis and for travel certification of young dogs. [ABSTRACT FROM AUTHOR]

Abstract: The assessment of fox immunity following oral rabies vaccination (ORV) is commonly applied to assess the efficacy of an ORV campaign in the field. Several ELISA kits have been developed and validated for their use for rabies serology in wildlife as an alternative to neutralizing techniques (NT), such as the fluorescent antibody virus neutralization test (FAVN) and the rapid fluorescent foci inhibition test (RFFIT). At a European level, NT and ELISA tests are used interchangeably and on different types of samples collected for vaccination follow-up. This has resulted in a difficulty in comparing the results generated with different diagnostic tools. We have evaluated (a) the effect of two different matrices commonly used for serology in red foxes on the results of the FAVN and (b) the performance of two commercially available ELISAs in comparison with the FAVN, as a gold standard, using a panel of over 700 field fox samples. Moderate agreement was observed when comparing results from different matrices. We found a very low level of agreement and low values of relative sensitivity and specificity of the ELISAs tested in comparison with the FAVN. Our findings confirm, using a vast collection of field samples obtained during post-vaccination surveillance campaigns in Italy, the need for improved reliability of certain serological tests. [Copyright &y& Elsevier]

During the last years the wild animals are vector and reservoir, and an important factor of the rabies in Lithuania. Analysis performed during the period from 2003 to 2007 by immunoflourescence (IF) had shown that 72.81% of raccoon dogs and 52.28% of foxes samples were positive for rabies. Oral vaccination (ORV of foxes with bites containing live rabies virus (Lysvulpen, Bioveta, Czech Republic) started in June 2006-September 2007. The present study investigated the effectiveness of ORV in Lithuania foxes considering that an antibody titer ≥0.5IU/mL is protective. During the study period 490 of foxes sera samples were tested by ELISA Platelia Rabies II test (Bio-Rad, France) and in 238 of tested samples (48.57%) were found antibodies for rabies. Furthermore, the comparison of 50 samples of fox sera using IF and fluorescent antibody virus neutralization (FAVN) test methods had shown comparable sensitivity. Some wildlife specimens were cytotoxic in the FAVN, resulting in possible false positive reaction. In view of this ELISA is rapid and simple technique on field fox sera. We obtained, with fox sera sampled in the same area, the same distribution of high, medium and low titres within all categories of serum quality (high to very poor quality) and therefore conclude that this ELISA test allows a reliable titration even with highly contaminated body fluids. The results demonstrated 92% correlation between IF and FAVN methods. [ABSTRACT FROM AUTHOR]

Abstract: Since 2000, there has been a legal requirement in the UK that dogs and cats should have an effective rabies vaccination with demonstrable sero-conversion if their owners wish to avoid quarantine on re-entry to the UK. In 2002, 10,483 rabies titres were determined on dogs at the VLA. Statistical analyses assessed the efficacy of each vaccine within different dog breeds. Animal size, age, breed, sampling time and vaccine had significant effects on pass rates and median titres. Our data suggests that a general relationship between animal size and level of antibody response exists and smaller sized dogs elicited higher antibody levels than larger breeds of dog. It was not however, only the magnitude of response immediately following vaccination but also the duration of immunity that varied between breeds of dog. Another observation was that young animals, less than 1-year of age, generated a lower antibody response to rabies vaccination than adults. Considerably higher failure rates were also observed for different vaccines tested. Regression analysis revealed that two vaccines performed equally well, and significantly better than the others tested. The variation in antibody response relating to length of interval of sampling following vaccination is not unexpected and presumably relates to the response kinetics for primary vaccination. These data need to be placed in perspective in order to minimise the risk of rabies being re-introduced into a rabies-free country, especially in the consideration of removing the requirement for serological testing for rabies vaccinated dogs that participate in pet travel schemes. [Copyright &y& Elsevier]