Caracterização genética e distribuição geográfica do vírus rábico isolado de bovinos no estado de Mato Grosso, Brasil / Genetic characterization and geographic distribution of rabies virus isolates from cattle in Mato Grosso, Brazil

Foram caracterizados, geneticamente e geograficamente, o sequenciamento parcial da nucleoproteína (gene N) de 53 isolados do vírus da raiva (VR) originários do Estado de Mato Grosso, Brasil. Os isolados de bovinos, que se encontravam no grupo do VR relacionado a morcegos hematófagos, foram posteriormente subdivididos em sete subgrupos genéticos. Estes subgrupos foram distribuídos em regiões de terras planas, com alguns subgrupos separados por formações de pequenas montanhas e hidrografia. Estes resultados indicam que a raiva em bovinos é derivada de diversas variantes regionalmente definidas, o que sugere que sua distribuição geográfica está relacionada as populações de morcegos hematófagos.

A total of 53 rabies virus (RV) isolates originating from cattle in the state of Mato Grosso, Brazil, 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 7 subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by small mountains and hydrographical features. 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.

A unique substitution at position 333 on the glycoprotein of rabies virus street strains isolated from non-hematophagous bats in Brazil.

The amino acid R or K at position 333 on the glycoprotein of the rabies virus is considered necessary for virulence in adult mice. Although some exceptions exist, substitution at this position causes expression of a phenotype that is either less pathogenic or non-virulent. To date, such substitutions have only been found in fixed strains of rabies virus. In this study, the authors found 333H, 333N, and 333Q substitutions at this position in rabies virus street strains isolated from non-hematophagous bats in Brazil. These strains showed pathogenicity and lethality on passage using adult mice with the intracerebral route and were confirmed rabies-positive by immunofluorescent assay. This suggests that these strains maintain virulence. Our findings indicate that rabies virus street strains with these substitutions exist in the field and may result in infection cycles.

Electrocardiographic parameters of captive lions (Panthera leo) and tigers (Panthera tigris) immobilized with ketamine plus xylazine.

Twenty-seven healthy captive lions (Panthera leo) and 13 healthy captive tigers (Panthera tigris) from São Paulo Zoo (Fundação Parque Zoológico de Sã Paulo, São Paulo, Brazil) collection were selected for this study. They were anesthetized with ketamine (10 mg/kg) combined with xylazine (1-2 mg/kg) for physical examinations, hematologic and serum chemical analysis and electrocardiogram recording. The main aim of this research was to gather initial information about normal electrocardiographic parameters of large felids. Standard P-QRS-T deflections on leads described for domestic carnivores were analyzed, and they did not greatly differ from those of large felids, taking into account the greater weight and corporal mass of large felids. Heart rate of lions ranged from 42 to 76 beats per minute (bpm). Heart rate of tigers ranged from 56 to 97 bpm. In both species, the most common rhythm detected was normal sinus rhythm followed by sinus arrhythmia; wandering pacemaker was also observed with normal sinus rhythm or sinus arrhythmia. Mean electrical axis lay between +60 degrees and +120 degrees. QRS complexes were predominantly positive in leads DI, DII, DIII, and AVF, and negative in AVR and AVL. This study provides insights into normal electrocardiograms of large felids. Wider investigations on the same subject are necessary to establish criteria for the recognition of abnormalities in these species and should include other anesthetic drug(s) combinations and reports of electrocardiographic features of animals with cardiac disease and electrolytes disturbances.

Genetic diversity of bat rabies viruses in Brazil.

Thirty-three Brazilian bat rabies viruses (RVs) were studied by sequence analysis and were compared against sequences of bat-related RVs from other regions of the Americas. Phylogenetic analysis revealed that bat-related RVs formed several monophyletic lineages and that these were associated with bat species. Brazilian bat RVs were found to include nine major lineages, one of which grouped with RVs isolated from Lasiurus spp. from different regions of the Americas. These results suggest that there is considerable diversity among Brazilian bat RV variants and that some of these RV variants may be associated with bats from other countries.

Molecular epidemiology of rabies from Maranhão and surrounding states in the northeastern region of Brazil.

Although many outbreaks of rabies have been reported in northern Brazil, few epidemiological studies of these outbreaks have been undertaken. In this study, molecular epidemiological analyses were performed using 41 rabies virus samples isolated in the Maranhão (MA), Pará (PA), and Tocantins (TO) states of northeastern Brazil. A 599-bp region of the glycoprotein (G) gene was first amplified from each sample by RT-PCR, then sequenced and subjected to phylogenetic analysis. A phylogenetic tree divided the 41 isolates into two clades: Clade I was associated with terrestrial carnivores and Clade II was associated with vampire bats. The Clade I isolates were further sub-divided into two groups. The first group was closer to carnivore isolates that predominate in central Brazil, whereas the second group more closely resembled wild fox isolates from the northeastern coastal state of Paraíba (PB). MA isolates of Clade II formed an entirely separate group. These results demonstrate that bat- and dog-transmitted rabies occur in northwestern Brazil.

Genetic and phylogenetic characterization of rabies virus isolates from wildlife and livestock in Paraiba, Brazil.

Thirty-four rabies virus (RV) isolates from foxes (8), insectivore bats (9), cattle (14), sheep (1), a goat (1) and a donkey (1) from Paraiba state, northeastern Brazil, were genetically characterized. Sequences of 890 nts of nucleoprotein (N) genes of these isolates were analyzed and compared with those of other Brazilian isolates characterized earlier. Phylogenetic analysis revealed three genetical lineages of RV co-existing in this region. Each lineage was found to be associated with particular host species and to circulate independently of each other. The first lineage was found in foxes (Dusicyon sp.) and could be discriminated from domestic carnivore isolates from Sao Paulo, Goias and Minas Gerais in the southern and central Brazil. The second lineage was associated with insectivorous bats (Molossus spp.) and differed from vampire bat-associated RV isolates. The third lineage was found in livestock and clustered with vampire bat-associated RV isolates from Sao Paulo, Tocantins, Goias and Matto Grosso. These results indicate that RV of these genetic lineages are cocirculating in the Paraiba state and that livestock in this region are infected with vampire bat-associated RV, suggesting that the vampire bat is the main reservoir of livestock rabies in this region.

Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir.

Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study.

Genealogical analyses of rabies virus strains from Brazil based on N gene alleles.

Thirty rabies virus isolates from cows and vampire bats from different regions of São Paulo State, Southeastern Brazil and three rabies vaccines were studied genetically. The analysis was based on direct sequencing of PCR-amplified products of 600 nucleotides coding for the amino terminus of nucleoprotein gene. The sequences were checked to verify their genealogical and evolutionary relationships and possible implication for health programmes. Statistical data indicated that there were no significant genetic differences between samples isolated from distinct hosts, from different geographical regions and between samples collected in the last two decades. According to the HKA test, the variability observed in the sequences is probably due to genetic drift. Since changes in genetic material may produce modifications in the protein responsible for immunogenicity of virus, which may eventually cause vaccine failure in herds, we suggest that continuous efforts in monitoring genetic diversity in rabies virus field strains, in relation to vaccine strains, must be conducted.

A heminested polymerase chain reaction for the detection of Brazilian rabies isolates from vampire bats and herbivores.

A heminested-PCR (hn-PCR) using primers to the nucleoprotein-coding gene in a nested set was evaluated in the detection of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein sequences belonging to genotype 1 were aligned. Based on such alignment, primers were directed to highly conserved regions. All 42 clinical samples positive by both fluorescent antibody and mouse inoculation tests were also positive by the hn-PCR. Brain tissue that had been left to decompose, obtained from an experimentally inoculated mouse was tested by hn-PCR and yielded positive results. In conclusion, primers designed here were capable of amplifying Brazilian RV isolates obtained from a rural epidemiological cycle.

A heminested polymerase chain reaction for the detection of Brazilian rabies isolates from vampire bats and herbivores

A heminested-PCR (hn-PCR) using primers to the nucleoprotein-coding gene in a nested set was evaluated in the detection of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein sequences belonging to genotype 1 were aligned. Based on such alignment, primers were directed to highly conserved regions. All 42 clinical samples positive by both fluorescent antibody and mouse inoculation tests were also positive by the hn-PCR. Brain tissue that had been left to decompose, obtained from an experimentally inoculated mouse was tested by hn-PCR and yielded positive results. In conclusion, primers designed here were capable of amplifying Brazilian RV isolates obtained from a rural epidemiological cycle

Genetic characterization and geographic distribution of rabies virus isolates in Brazil: identification of two reservoirs, dogs and vampire bats.

We analyzed 50 rabies virus samples isolated in Brazil from 12 dogs, 11 cats, 5 vampire bats, 15 cattle, 2 horses, 1 pig, 1 sheep, and 3 humans to investigate the molecular epidemiology of rabies viruses. We sequenced 203 nucleotides on the nucleoprotein gene by direct sequencing of the PCR-amplified products. All the isolates belonged to the genotype 1 and homology of the 203 nucleotides was at least 83.7% among isolates. The main reservoirs were estimated based on the homology of nucleotide sequences. Brazilian rabies virus isolates were clustered into two reservoir groups: dogs and vampire bats. All the dog-related rabies virus isolates showed nucleotide homology greater than 99.0%. Vampire bat-related rabies virus isolates showed nucleotide homology greater than 96.6% and could be further divided into subgroups corresponding to areas where viruses were isolated. These data suggest that circulating rabies variants belong to at least two different genotype clusters in Brazil and that these two clusters are maintained independently among vampire bats and dogs.

Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference.

Maternal antibody passively transferred interferes with rabies vaccination in hamsters.

Transference and interference of maternal immunity to offspring after rabies vaccination were studied in hamsters. Females were vaccinated or not before mating and offspring were vaccinated at the age of 7, 14, 21 and 30 days. Other pups were maintained as controls. Thirty days after vaccination pups were challenged intracerebrally with CVS virus. Mouse neutralization tests were used to verify antibody titers. Mortality of 97.0, 76.9, 60.9 and 24.0% was observed in pups vaccinated at 7, 14, 21 and 30 days respectively, born from vaccinated dams, while in pups from non-vaccinated dams, mortality was 51.4, 28.6, 8.7 and 0.0%. Statistically significant associations were found between mortality and age at vaccination, by simple linear regression with y=-3.1169x + 120.8 (p = 0.008; r2=0.98) for litters vaccinated and born from vaccinated dams and y=-2.2541x + 62.7495 (p = 0.03; r2=0.93) for pups vaccinated and born from non-vaccinated dams. Immunological response to vaccination in pups born from vaccinated mothers was delayed 11 days, when compared to that observed in pups of non-vaccinated mothers.

Effect of the bacillus of Calmette-Guérin, Propionibacter acnes and avridine as immunomodulators in antirabies vaccination of mice using the Fuenzalida-Palacios mouse brain vaccine.

Using the laboratory mice, Fuenzalida-Palacios mouse brain human rabies vaccine was administered in groups of animals previously inoculated with rabies virus and then submitted to treatments with the immunomodulators onco-BCG, avridine and Propionibacterium acnes. Humoral and cellular immune responses were evaluated through the macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). The IPI test was not effective in detecting the response of delayed-type hypersensitivity, contrary to MIF, which showed the immune cellular response. Higher levels of IFN-gamma were observed in the groups of mice vaccinated and treated with avridine and P. acnes. Although immunomodulating activities have been detected, the use of adjuvants with the Fuenzalida-Palacios type vaccine in mice did not reveal any encouraging results.

Effect of bacillus of Calmette-Guérin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice.

The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-gamma (IFN-gamma) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-gamma concentration and MIF response.

Effect of bacillus of Calmette-Guerin, avridine and Propionibacterium acnes as immunomodulators on rabies in mice

Avaliou-se a resposta imune celular e humoral de camundongos inoculados com virus rabico 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 sobrevivencia quando comparados aos dos demais tratamentos. Foram observados menores niveis de IFN-gama nos animais infectados, sugerindo imunossupressao viral. O teste do Coxim Plantar nao foi eficaz para a deteccao da resposta de hipersensibilidade retardada na metodologia utilizada, contrariamente ao MIF. A sobrevivencia dos animais nao apresentou correlacao com os niveis de anticorpos soroneutralizantes, concentracao de IFN-gama e resposta ao MIF

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.

Effect of vaccination and the immunomodulators "bacillus of Calmette-Guérin, avridine and Propionibacterium acnes" on rabies in mice.

Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma). Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice. Higher survival rates were found in animals treated with P. acnes. Lower levels of IFN-gamma were observed in the groups of infected and vaccinated mice. The IPI was not effective on detecting the response of delayed-type hypersensitivity. Vaccine induced in the infected animals a more intense response to MIF reaction.

Effect of a booster-dose of rabies vaccine on the duration of virus neutralizing antibody titers in bovines.

Humoral immune response using inactivated rabies vaccine was studied in 35 nelore cross-bred bovines of western region of São Paulo state. Ninety days after vaccination, 13 (92.8%) animals presented titers > or = 0.5 IU/ml, through mouse neutralization test. After 180 days, 9 (64.3%) sera showed titers > or = 0.5 IU/ml, after 270 days, only one (7.1%) showed a titer of 0.51 IU/ml, and after 360 days, all animals showed titers < 0.5 IU/ml. Group of animals receiving booster dose 30 days after vaccination presented, two months after, all with titers > 0.5 IU/ml. At 180 days, 17 (80.9%) sera presented titers > 0.5 IU/ml; at 270 days, 15 (71.4%), with titers > or = 0.5 IU/ml and at 360 days, 4 (19.0%), with titers > or = 0.5 IU/ml. Booster-dose ensured high levels of neutralizing antibodies for at least three months, and 240 days after revaccination, 71.4% of animals were found with titers > or = 0.5 IU/ml.

Leptospira interrogans serovar icterohaemorrhagiae seropositivity and the reproductive performance of sows.

The reproductive performance of 28 sows seropositive to Leptospira interrogans serovar icterohaemorrhagiae was compared with that of 87 Leptospira sp. seronegative dams belonging to the same herd. Sows were sampled during 1988 to 1993. During this period the herd was not submitted to any kind of intervention (antibiotic therapy, immunoprophylaxis or rodent control). Relative risks (RR) of return to heat, mummified fetuses, stillbirth, and weak newborn piglets for infected sows were assessed and the differences in means of total piglets born per litter, piglets born alive, piglets effectively housed, weaned piglets, stillbirths, mummified fetuses, weak newborn piglets, weight at birth of the piglets effectively housed, weight at 21 days of life and weight at weaning were evaluated. Seropositive dams had a greater risk of having weak newborn piglets (RR = 1.67, 1.02 < or = CI 95% < or = 2.72) and also of having more weak newborn piglets per litter (P = 0.01). Other variables examined were not different (P > 0.05).