Sidnei Miyoshi Sakamoto

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses.

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.

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.

Análise de tratamento anti-rábico humano pós-exposição em região da Grande São Paulo, Brasil

Abstract Introduction The treatment of human beings with risk of exposure to rabies virus infection stillpresents some cases of undesirable post treatment reactions. The knowledge re-sulting from the regular analysis of the epidemiological features associated withthese practices would minimize the occurrence of these troubles. Methods The records of the treatment of 8,758 human anti-rabies cases performed inOsasco City, S. Paulo, Brazil between 1984 and 1994 with Fuenzalida & Palaciosvaccine and/or anti-rabies serum produced in equines were analysed by Epi Infoprogram. Results It was found that the higher risk of exposure was for male children of from five tonine years of age. The most frequent scene of aggression was the victim’s ownhouse. Dogs were responsible for most of the accidents (62.8%); the majority ofdogs and cats involved had not been immunized against rabies (51.0% of dogs and73.2% of cats). Most people aged nine or under were attacked in the head(36.6%), followed by the upper limbs (35.1%); above this age the part of the bodymost frequently hurt was the upper (45.8%) and lower limbs (43.7%); 26.5% ofpatients had already been vaccinated against rabies at least once in their lifetime;90.7% of them sought medical care within five days, anti-rabies treatment wasprescribed for 41.9% of them.INTRODUCTION: The treatment of human beings with risk of exposure to rabies virus infection still presents some cases of undesirable post treatment reactions. The knowledge resulting from the regular analysis of the epidemiological features associated with these practices would minimize the occurrence of these troubles. METHODS: The records of the treatment of 8,758 human anti-rabies cases performed in Osasco City, S. Paulo, Brazil between 1984 and 1994 with Fuenzalida & Palacios vaccine and/or anti-rabies serum produced in equines were analysed by Epi Info program. RESULTS: It was found that the higher risk of exposure was for male children of from five to nine years of age. The most frequent scene of aggression was the victims own house. Dogs were responsible for most of the accidents (62.8%); the majority of dogs and cats involved had not been immunized against rabies (51.0% of dogs and 73.2% of cats). Most people aged nine or under were attacked in the head (36.6%), followed by the upper limbs (35.1%); above this age the part of the body most frequently hurt was the upper (45.8%) and lower limbs (43.7%); 26.5% of patients had already been vaccinated against rabies at least once in their lifetime; 90.7% of them sought medical care within five days, anti-rabies treatment was prescribed for 41.9% of them. CONCLUSIONS: Post vaccinal reactions were observed in 0.25% of the patients 0.3% of which were of neurological; 80.1% of the dogs and 58.4% of the cats involved coere kept under observation. The months of March, July, August and September presented the highest aggression rates.

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.

Taxonomic Status of Ixodes didelphidis (Acari: Ixodidae)

Abstract Ixodes didelphidis Fonseca & Aragão was described in Brazil in 1952 as a new tick species that differed from Ixodes loricatus Neumann by the spiracular plate pattern. We have reared four tick colonies from different geographic areas in the laboratory that were started from single engorged females originally identified as I. didelphidis (BMG colony) and I. loricatus (CSP, PSP, and TRJ colonies). We analyzed the spiracular plate morphology of F1 adult ticks from each tick colony, compared their biological data, and performed a molecular analysis of the second internal transcribed rDNA spacer (ITS2) to test the validity of the species I. didelphidis. The spiracular plate analysis of laboratory F1 adult ticks yielded from single females from the four colonies showed variations that invalidate morphological parameters for differentiation of I. loricatus and I. didelphidis. Biological data of the BMG, CSP, and TRJ colonies were similar. The biology of the PSP colony was not evaluated. The ITS2 sequence variations observed between the tick colonies ranged from 1.3 to 4.9%, and the similarity tree constructed by the neighbor-joining method with nucleotide distances showed that the distances between the samples were similar to what is expected for intraspecific variations found in other ticks species. The morphological and biological results, in conjunction with the ITS2 analysis, supported the conspecificity of I. loricatus and I. didelphidis. RESUMO Ixodes didelphidis Fonseca e Aragão foi descrito no Brazil em 1952 como uma nova espécie de carrapato que se diferenciava de Ixodes loricatus Neumann através do padrão da placa espiracular. Nós criamos em laboratório quatro colônias de carrapatos de diferentes áreas geográficas, iniciadas a partir de fêmeas ingurgitadas originalmente identificadas como I. didelphidis (colônia BMG) e I. loricatus (colônias CSP, PSP, e TRJ). Nós analisamos a morfologia da placa espiracular dos adultos F1 de cada colônia, comparamos seus dados biológicos e fizemos uma análise molecular do segundo espaço transcrito interno do rDNA (ITS2) para verificar a validade da espécie I. didelphidis. A análise da placa espiracular de adultos provenientes de uma mesma fêmea de cada uma das colônias demonstrou resultados controversos que invalidam os parâmetros morfológicos publicados para diferenciação de I. loricatus e I. didelphidis. Os dados biológicos das colônias BMG, CSP, e TRJ foram similares. As variações nas seqüências do ITS2 entre as colônias de carrapatos (amplitude: 1,3–4,9%) e a árvore de similaridade com as distâncias de nucleotídeos mostraram que as distâncias entre as amostras foram similares ao esperado para variações intra-específicas encontradas em outras espécies de carrapatos. Os resultados morfológicos, biológicos e moleculares fornecem evidências para considerarmos I. loricatus e I. didelphidis como a mesma espécie.

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).