Fernanda Bernardi

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.

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.

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.

Isolation of rabies virus from the parotid salivary glands of foxes (Pseudalopex vetulus) from Paraíba State, Northeastern Brazil.

To determine the presence of rabies virus in the parotid salivary glands, 12 road-killed rabies-positive hoary foxes (Pseudoalopex vetulus) were tested by using the fluorescent antibody test (FAT) and mouse inoculation test (MIT). All 12 parotid salivary glands were positive for both tests, although in some cases several passages were required. The findings of this study support the importance of the hoary fox as rabies reservoir in the sem-iarid region of Paraíba State, Northeastern Brazil.

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.

Aplicação experimental de um modelo de conduta de inspeção sanitária no comércio varejista de alimentos

The aim of this research was to apply a diagnostic procedure and a model of sanitary inspection in food establishments in the city of Ibiuna, State of Sao Paulo, Brazil. Results were analyzed in order to achieve sanitary surveillance goals. The work was developed during six months in 19 retail food establishments such as bakeries, restaurants, and butcheries. Firstly, the establishment situation was diagnosed; irregularities were verified according to hygienic-sanitary conditions. An average 64 points was obtained. The next step included new and frequent sanitary surveillance team visits to those establishments in order to verify their sanitary quality adequacies. Inspection guides were used and detailed reports were prepared for the establishment owners. Those who handled food were trained in basic food hygiene. Results indicate that after 3 inspection visits to each establishment, 100% of them presented satisfactory results with 90 point average. This sanitary inspection model is considered useful to promote food safety.

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.

Prevalência e fatores de risco para a leptospirose e brucelose na população canina da Estância Turística de Ibiúna, São Paulo, Brasil

The aim of this survey was to determine the prevalence of leptospirosis and brucellosis due to Brucella canis and to determine the risk factors associated with positivity in dogs of the Tourist Resort of Ibiuna, State of Sao Paulo, Brazil. A total of 570 blood samples were collected from dogs from 4 regions of 48 districts of the county of Ibiuna during the period of September 2007 to March 2008. Serological diagnosis of leptospirosis was performed with the microscopic agglutination test (MAT), and blood culture was used for the diagnosis of brucellosis. Of the 570 dogs used 187 (32.8%; 95%CI 28.9 - 36.8) were seropositive to leptospirosis, with predominance of reactions to serovars Pyrogenes, Autumnalis and Canicola, and 6 (1.05%; 95%CI 0.4 - 2.2) were positive to brucellosis. Variable sexual activity (OR = 1.73) was identified as risk factor associated with the positivity to leptospirosis, and free access to street was considered risk factor for both leptospirosis (OR = 1.96) and brucellosis (OR = 10.85). It is concluded that leptospirosis and brucellosis are present in dogs of the Tourist Resort of Ibiuna, State of Sao Paulo, and sexual activity and free access to street are conditions associated with the prevalence of infections.