Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Rabies infection and specific effect of vaccination in mice selected for high and low immunobiological parameters

Innate and acquired resistance to rabies infection was investigated in mice genetically selected for high (H) or low (L) antibody responsiveness from selections I, III and IV and in mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction. These mouse lines were infected intramuscularly with different virus dilutions and the LD50 was determined. The HIII and HIV mouse lines were more susceptible than the LIII and LIV lines and the HI line showed a discrete but higher resistance than the LI line. Analysis of the interline (H x L) F1 hybrids from selections III and IV indicated different dominance effects on the resistant and susceptible phenotypes when the route of vaccination was changed. No differences were observed between the AIRmax and AIRmin mice, suggesting that inflammation plays a minor role in the resistance to rabies virus. The comparison of LD50 in mice vaccinated by distinct routes showed that the highest interline difference occurred after intramuscular vaccination (250-fold between H and L and 800-fold between F1 and L). These results indicate that different mechanisms may participate in acquired antirabies resistance

Bat rabies in the north-northwestern regions of the state of São Paulo, Brazil: 1997-2002

OBJETIVO: Os relatos sobre a ocorrência de raiva em morcegos no Brasil são esporádicos e isolados. Assim, o objetivo do estudo foi descrever a detecção do vírus da raiva em morcegos do Estado de São Paulo. MÉTODOS: Foram analisados 7.393 morcegos provenientes de 235 municípios do norte e noroeste do Estado de São Paulo, no período de 1997 a 2002 e identificados por meio de características morfológicas e morfométricas. Para a detecção do antígeno viral foi utilizada a técnica de imunofluorescência direta e o isolamento do vírus foi realizado por inoculação em camundongos. RESULTADOS: Das amostras examinadas, 1,3% foram positivas para raiva, com variação de 0,2% em 1997 a 1,6% em 2001. Foram encontrados 98 morcegos com o vírus, 87 deles em área urbana. O vírus da raiva foi detectado pela imunofluorescência direta em 77 do total de amostras positivas, enquanto 92 produziram doença em camundongos inoculados e o período de incubação variou entre 4-23 dias. em 43 municípios foi encontrado pelo menos um morcego positivo. Entre as espécies analisadas o vírus da raiva foi detectado com maior freqüência (33,7%) em Artibeus lituratus. Os vespertilionideos do gênero Eptesicus e Myotis totalizaram 24,5% dos morcegos positivos e as espécies do gênero Molossus (Molossus molossus e Molossus rufus), 14,3%. A distribuição do vírus da raiva foi semelhante entre fêmeas (33; 48,5%) e machos (35; 51,5%). CONCLUSÕES: Morcegos positivos para raiva foram encontrados em situações que colocam em risco tanto a população humana como animais de estimação, exigindo medidas voltadas para o manejo destas espécies e de educação da população.

Effect of Bacillus of Calmette-Guérin, Avridine and Propionlbacterium acnes as immunomodulators on rabies in mice

Avaliou-se a resposta imune celular e humoral de camundongos inoculados com vírus rábico de rua e submetidos aos imunomoduladores Onco-BCG, avridina e Propionibacterium acnes. Os animais submetidos ao tratamento com P.acnes apresentaram um maior percentual de sobrevivência quando comparados aos dos demais tratamentos. Foram observados menores níveis de IFN-g nos animais infectados, sugerindo imunossupressão viral. O teste do Coxim Plantar não foi eficaz para a detecção da resposta de hipersensibilidade retardada na metodologia utilizada, contrariamente ao MIF. A sobrevivência dos animais não apresentou correlação com os níveis de anticorpos soroneutralizantes, concentração de IFN-g e resposta ao MIF.

Genetic analysis of phosphoprotein and matrix protein of rabies viruses isolated in Brazil

To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with ASP(42) and GlU(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants.

Geographical distribution of vampire bat-related cattle rabies in Brazil

Seventy-seven rabies virus (RV) isolates originating from Brazilian cattle were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat-related RV group, were further subdivided into nine genetic subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by mountain ranges. In addition, separation of the groups in mountainous regions was correlated with altitude. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.

Potential force of infection of human rabies transmitted by vampire bats in the Amazonian region of Brazil

Human rabies tansmitted by bats has acquired greater epidemiologic relevance in various Latin American countries, just when cases transmitted by dogs have decreased. Concern has been heightened by reports of increased rates of bats biting humans in villlages in the Amazonian region of Brazil. The aim of the present work was to estimate the potential force of infection (per capita rate at which susceptible individuals acquire infection) of human rabies transmitted by the common vampire bat if the rabies virus were to be introduced to a colony of bats close to a village with a high rate of human bites. The potential force of infection could be then used to anticipate the size of a rabies outbreak in control programs. We present an estimator of potential incidence, adapted from models for malaria. To obtain some of the parameters for the equation, a cross-sectional survey was conducted in Mina Nova, a village of gold prospectors in the Amazonian region of Brazil with high rates of bates biting humans. Bats were captured near dwellings and sent to the Rabies Diagnostic Laboratory at the Center for Control of Zoonoses (São Paulo, Brazil) to be examined. To estimate the force of infection, a hypothetical rabies outbreak among bats was simulated using the actual data obtained in the study area. of 129 people interviewed, 23.33% had been attacked by a vampire bat during the year prior to the study, with an average of 2.8 bites per attacked person. Males (29.41%) were attacked more often than females (11.36%); also, adults (29.35%) were attacked more often than children (8.33%). None of the 12 bats captured in Mina Nova tested positive for rabies, but the force of infection for a hypothetical outbreak was estimated to be 0.0096 per person per year. This risk represents 0.96 cases per 100 area residents, giving an incidence of 1.54 cases of bat-transimtted buman rabies per year in the village of Mina Nova (160 inhabitants). The estimated risk is comparable with what has been observed in similar Brazilian villages.

Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil

In Latin America, rabies cases related to frugivorous bats have been reported since 1930's. Recently, two viruses isolated from Artibeus lituratus were proved to be vampire bat variants by monoclonal antibodies panels [2], but their genetic information is not well known. In this report, four rabies viruses were isolated from frugivorous bats (Artibeus spp.) in Brazil and their nucleoprotein gene sequences were determined. These isolates were found to be genotype 1 of lyssavirus and showed the maximum nucleotide sequence homology of 97.6-99.4% with vampire bat-related viruses in Brazil [6]. These results indicate that the Brazilian frugivorous bat rabies viruses in this study are closely related to vampire bat-related viruses that play a main role in rabies virus transmission to livestock in Brazil.

Herpes Virus Bovino tipo 1 (BoHV-1) como posible causa de encefalitis en bovinos de la región del Magdalena Medio Colombiano. Estudio serológico y análisis epidemiológico

The bovine Herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5), causing diseases and significant economic losses in farms of worldwide. Both affect the nervous system of cattle, although BoHV-5 has been the most associated with this type of pathogenesis. Given the death of animals with nervous symptoms and negative diagnoses for rabies virus in the area of study, this research focused on the detection of positive reactors to bovine herpes virus serum neutralization. We collected 518 blood samples from animals without Herpesvirus vaccine, in the municipalities of Caparrapi, Cimitarra, Honda and Victoria, in the Middle Magdalena River Region. In addition, epidemiological information useful to discuss neurological disease was collected through primary and secondary sources. For the analysis of data was used chi-square test by identification of relationship between evidence of viral infection and the variables recorded. The results revealed that 286 cases were positive for Herpesvirus infection, corresponding to a prevalence of 55.5%, however, there was no statistical relationship (p < 0.05) between the presence of antibodies and the variables analyzed. In conclusion, some cases of neurological disease in cattle in this region could be due to infection with herpes viruses. We discussed about the presence of BoHV-1 and BoHV-5 in the ambient, diagnosis and monitoring plans, as well as economic losses, which may cause in herds in this area.

Rabies in southeast Brazil: a change in the epidemiological pattern

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background: Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.Methods: Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.Results: In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.Conclusion: This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

Influence of canine brain decomposition on laboratory diagnosis of rabies.

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29 degrees C for up to 168 h. At 24 h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72 h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48 h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.

Antibody and cytokine serum levels in patients subjected to anti-rabies prophylaxis with serum-vaccination

Rabies is considered a fatal disease once clinical symptoms have developed. The aim of this study was to evaluate epidemiological aspects and immune response in patients attacked by domestic and wild animals and subjected to post-exposure rabies treatment with equine serum and associated vaccine. Thirty-three patients were evaluated; they were between 13 and 65 years old, 75.8% were male and 24.2% female, and from the Botucatu neighborhood. Twenty healthy control individuals with the same age range were also studied. Specific antibodies to equine immunoglobulins and IFN-γ, IL-2, IL-4, and IL-10 production were evaluated by ELISA. IgM, IgE, IgG and subclasses, and rabies virus antibodies serum levels were determined by nephelometry and seroneutralization methods, respectively. No anaphylactic or serum sickness allergic reactions were observed in patients after treatment. Anti-equine IgG levels were significantly higher than those of IgM after 14 and 28 days of treatment. Protective antibodies to rabies virus > 0.5 UI/ml were detected in 84.6% and 75% of patients at days 14 and 28, respectively. IFN-γ, IL-2 and IL-10 levels in patients before and 48h after treatment were significantly higher than in controls suggesting that both Th1 and Th2 cells were activated in the patients. Serum IgM levels were higher at day 14, and IgG 2 and IgE levels were higher at day 28 of treatment. These results suggest that post-exposure rabies treatment in humans induces significant alterations in patient immune response characterized by increased levels of cytokines, serum levels of specific rabies virus antibodies, and the equine serum components employed in the treatment.

Rabies surveillance in bats in Northwestern State of Sao Paulo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Landscape-Genetic Analysis of Population Structure in the Texas Gray Fox Oral Rabies Vaccination Zone

In west-central Texas, USA, abatement efforts for the gray fox (Urocyon cinereoargenteus) rabies epizootic illustrate the difficulties inherent in large-scale management of wildlife disease. The rabies epizootic has been managed through a cooperative oral rabies vaccination program (ORV) since 1996. Millions of edible baits containing a rabies vaccine have been distributed annually in a 16-km to 24-km zone around the perimeter of the epizootic, which encompasses a geographic area >4 x 105 km2. The ORV program successfully halted expansion of the epizootic into metropolitan areas but has not achieved the ultimate goal of eradication. Rabies activity in gray fox continues to occur periodically outside the ORV zone, preventing ORV zone contraction and dissipation of the epizootic. We employed a landscape-genetic approach to assess gray fox population structure and dispersal in the affected area, with the aim of assisting rabies management efforts. No unique genetic clusters or population boundaries were detected. Instead, foxes were weakly structured over the entire region in an isolation by distance pattern. Local subpopulations appeared to be genetically non-independent over distances >30 km, implying that long-distance movements or dispersal may have been common in the region. We concluded that gray foxes in west-central Texas have a high potential for long-distance rabies virus trafficking. Thus, a 16-km to 24-km ORV zone may be too narrow to contain the fox rabies epizootic. Continued expansion of the ORV zone, although costly, may be critical to the long-term goal of eliminating the Texas fox rabies virus variant from the United States.