A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations. (C) 2007 Elsevier B.V. All rights reserved.

Comparison of infection by Brucella spp. in free-ranging and captive wild animals from São Paulo State, Brazil

The aim of the current study was to evaluate the infection rate by Brucella spp. in wild and in captive animals. Serum samples from 121 animals (94 free-ranging and 27 captive) of different mammal species were evaluated. Sera were submitted to rose Bengal test (RBT) for screening and serum agglutination tests (SAT) and 2-mercaptoethanol test (2-ME) for confirmatory results. Nine animals (five free-ranging and four captive) tested positive in RBT, but negative in the confirmatory tests. Several domestic animal diseases that have control programs are not focused on wild reservoirs, such as brucellosis in Brazil. The study of new reservoirs in wildlife is essential to prevent emerging diseases.

Hantaviruses as emergent zoonoses

Hantaviruses belong to the Bunyaviridae family, which consists of vector-borne viruses. These viruses can provoke two infection types: hemorrhagic fever with renal syndrome (HFRS) - which occurs in the Old World - and hantavirus cardiopulmonary syndrome (HCPS) - an emergent zoonosis that can be found in many countries of the western hemisphere. Rodents are hantavirus reservoirs and each species seems to host a different virus type. Humans acquire the infection by inhaling contaminated aerosol particles eliminated by infected animals. The factors involved in the emergence of hantavirus infections in the human population include ecological modifications and changes in human activities. The most important risk factor is contact between man and rodents, as a result of agricultural, forestry or military activities. Rodent control remains the primary strategy for preventing hantavirus diseases, including via health education and hygienic habits.

Road-killed wild animals: a preservation problem useful for eco-epidemiological studies of pathogens

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

Interactions between environment, wild animals and human leptospirosis

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

Brazilian spotted fever: A reemergent zoonosis

Brazilian spotted fever is caused by the bacterium Rickettsia rickettsii, which is the most pathogenic species of the spotted-fever rickettsiae group and is transmitted by the bite of infected ticks. Amblyomma cajennense is the most important tick species involved in the cycle of this zoonosis in Brazil as it presents low host specificity, great number of natural reservoirs and wide geographic distribution. It was first described in the state of São Paulo in 1929 and later in Rio de Janeiro, Minas Gerais and Bahia. The number of cases decreased in the 1940's with the development of new plague control techniques and antibiotics. In the last decades, the number of new cases has increased. The current review aimed at reporting some of the epidemiological and public health aspects of this reemergent disease with new foci, mainly in the southeastern region of Brazil.

SEROLOGIC SURVEY FOR LEPTOSPIRA SPP. IN CAPTIVE NEOTROPICAL FELIDS IN FOZ do IGUACU, PARANA, BRAZIL

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

Rabies virus distribution in tissues and molecular characterization of strains from naturally infected non-hematophagous bats

Bats are main reservoirs for Lyssavirus worldwide, which is an important public health issue because it constitutes one of the big challenges in rabies control. Yet, little is known about how the virus is maintained among bats, and the epidemiological relationships remain poorly understood. The aim of the present study was to investigate the distribution of the rabies virus (RABV) in bat tissues and organs and to genetically characterize virus isolates from naturally infected non-hematophagous bats. The heminested reverse transcriptase polymerase chain reaction (hnRT-PCR) and sequencing using primers to the nucleoprotein coding gene were performed. The results showed a dissemination of the RABV in different tissues and organs, particularly in the salivary glands, tongue, lungs, kidneys, bladder, intestine and feces, suggesting other possible forms of RABV elimination and the possibility of transmission among these animals. The phylogenetic analysis confirmed that different variants of RABV are maintained by non-hematophagous bats in nature and have similar tissue distribution irrespective of bat species and phylogenetic characterization. (C) 2012 Elsevier B.V. All rights reserved.

Spatial Distribution of Seropositive Dogs to Leptospira spp. and Evaluation of Leptospirosis Risk Factors Using a Decision Tree

Background: Leptospirosis is an important zoonotic disease associated with poor areas of urban settings of developing countries and early diagnosis and prompt treatment may prevent disease. Although rodents are reportedly considered the main reservoirs of leptospirosis, dogs may develop the disease, may become asymptomatic carriers and may be used as sentinels for disease epidemiology. The use of Geographical Information Systems (GIS) combined with spatial analysis techniques allows the mapping of the disease and the identification and assessment of health risk factors. Besides the use of GIS and spatial analysis, the technique of data mining, decision tree, can provide a great potential to find a pattern in the behavior of the variables that determine the occurrence of leptospirosis. The objective of the present study was to apply Geographical Information Systems and data prospection (decision tree) to evaluate the risk factors for canine leptospirosis in an area of Curitiba, PR.Materials, Methods & Results: The present study was performed on the Vila Pantanal, a urban poor community in the city of Curitiba. A total of 287 dog blood samples were randomly obtained house-by-house in a two-day sampling on January 2010. In addition, a questionnaire was applied to owners at the time of sampling. Geographical coordinates related to each household of tested dog were obtained using a Global Positioning System (GPS) for mapping the spatial distribution of reagent and non-reagent dogs to leptospirosis. For the decision tree, risk factors included results of microagglutination test (MAT) from the serum of dogs, previous disease on the household, contact with rats or other dogs, dog breed, outdoors access, feeding, trash around house or backyard, open sewer proximity and flooding. A total of 189 samples (about 2/3 of overall samples) were randomly selected for the training file and consequent decision rules. The remained 98 samples were used for the testing file. The seroprevalence showed a pattern of spatial distribution that involved all the Pantanal area, without agglomeration of reagent animals. In relation to data mining, from 189 samples used in decision tree, a total of 165 (87.3%) animal samples were correctly classified, generating a Kappa index of 0.413. A total of 154 out of 159 (96.8%) samples were considered non-reagent and were correctly classified and only 5/159 (3.2%) were wrongly identified. on the other hand, only 11 (36.7%) reagent samples were correctly classified, with 19 (63.3%) samples failing diagnosis.Discussion: The spatial distribution that involved all the Pantanal area showed that all the animals in the area are at risk of contamination by Leptospira spp. Although most samples had been classified correctly by the decision tree, a degree of difficulty of separability related to seropositive animals was observed, with only 36.7% of the samples classified correctly. This can occur due to the fact of seronegative animals number is superior to the number of seropositive ones, taking the differences in the pattern of variable behavior. The data mining helped to evaluate the most important risk factors for leptospirosis in an urban poor community of Curitiba. The variables selected by decision tree reflected the important factors about the existence of the disease (default of sewer, presence of rats and rubbish and dogs with free access to street). The analyses showed the multifactorial character of the epidemiology of canine leptospirosis.

Epidemiological aspects in leptospirosis. Research of anti-Leptospira spp antibodies, isolation and biomolecular research in bovines, rodents and workers in rural properties from Botucatu, SP, Brazil.

Leptospirosis is a worldwide infection, transmitted between man and animals that causes a decrease in the production of bovine flocks, and offer risks for public health, as an important zoonosis. The rodents are the main reservoirs of leptospires. It was studied 27 dairy farm properties located in or near from Botucatu-SP, Brazil. In these farms were collected blood and kidney samples from rodents, blood and urine samples from bovines and blood samples from the workers. The serology was performed with microscopic agglutination test (MAT). Samples of bovine urine and rodent kidneys were cultivated searching for leptospires isolation. The polymerase chain reaction (PCR) of the kidneys of the rodents was performed. In MAT, 46/ 140 (32.85%) bovine and 8/34 (23.53%) human sera samples were positive, respectively. In human samples, the serovar Brastilava (37.51%) presented the highest occurrence, while in bovines, the serovars Hardjo and Castellonis were most frequent, with 26.08% each one. All of the rodents were negatives in serology. No leptospire was isolated, and kidney samples were negative in PCR. In bovines, the dam water and the bad hygiene quality of milking process were considered important risks of infection in the affected properties (p<0.05), where other reproductive problems, except abortion, can be related. In other side, to human beings the drainage system was the most important risk factor in the studied properties. Thus, it was verified the necessity of an improvement in zoosanitary handling of the properties, mainly of water supply.

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

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

Evaluation of TNF-alpha, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-alpha, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-alpha, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-alpha showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

Presence of bacteria in dentinal tubules

This study demonstrated that a significant number of bacteria is present. in the radicular dentinal tubules of periodontally diseased human teeth. Ten periodontally diseased teeth were prepared and stained by Brown and Brenn technique for histological examination. Bacteria were detected in all teeth. It is suggested that bacteria may invade dentinal tubules exposed to periodontal pocket and are very hard to be eliminated by conventional mechanical and chemical periodontal therapy. Contaminated dentinal tubules of periodontally diseased teeth can thus act as active bacterial reservoirs to promote recolonization of mechanically treated root surfaces, which could interfere with the periodontal healing and progression of the disease.

Alterações anatômicas foliares em plantas de Brachiaria subquadripara submetidas à aplicação de herbicidas

O presente trabalho teve por objetivo avaliar a eficiência do controle dos herbicidas diquat, glyphosate e imazapyr e as alterações anatômicas do limbo foliar provocadas por eles em plantas de Brachiaria subquadripara. As plantas foram cultivadas em caixas-d'água sob condições de campo e, quando atingiram estádio de pleno desenvolvimento vegetativo, foram pulverizadas com soluções de diquat a 400 g ha-1, glyphosate a 4.320 g ha-1 em associação com o adjuvante Aterbane a 0,5% v v-1 e imazapyr a 750 g ha-1. Além disso, utilizou-se uma testemunha sem aplicação de herbicida. Foram realizadas avaliações de controle e das seguintes características anatômicas quantitativas das regiões da nervura central e da internervural do limbo foliar, além da região do colmo: porcentagem da epiderme adaxial e abaxial, porcentagem da endoderme, porcentagem do feixe vascular, porcentagem de lacunas do aerênquima, porcentagem de parênquima, espessura foliar, porcentagem da epiderme do colmo, porcentagem do câmbio vascular e diâmetro do colmo. Os herbicidas diquat e imazapyr promoveram o maior número de alterações nos caracteres anatômicos quantitativos das regiões da nervura central e internervural do limbo foliar e da região do colmo das plantas. Contudo, com exceção do diquat, os herbicidas foram eficientes no controle das plantas de B. subquadripara, porém todos eles permitiram a rebrota delas.

Diferenciação entre Egeria densa e Egeria najas pelos caracteres anatômicos foliares

Dentre as plantas daninhas aquáticas imersas de maior importância nos reservatórios de usinas hidrelétricas e em represas de pequeno porte no Brasil, destacam-se as espécies Egeria densa e Egeria najas, cuja identificação pode ser difícil na fase vegetativa. O objetivo deste trabalho foi diferenciar cinco acessos de E. densa e três de E. najas, coletados nos reservatórios de Jupiá, Salto Grande, Três Irmãos, Promissão, Nova Avanhandava e Ibitinga, do complexo da Companhia Energética de São Paulo (CESP) do Estado de São Paulo, quanto às características anatômicas descritivas e quantitativas do limbo foliar, procurando-se obter melhor entendimento sobre as relações dessas estruturas anatômicas com a penetração e translocação de herbicidas, além de auxiliar na identificação de acessos suscetíveis e resistentes a determinado produto químico. Amostras do terço médio do limbo foram fixadas em FAA 50, cortadas transversalmente em micrótomo rotatório com 8 mm de espessura e coradas com azul-de-toluidina. Foi analisada a estrutura foliar e foram quantificados os caracteres anatômicos da nervura central (% epiderme das faces adaxial e abaxial, % feixe vascular e % parênquima) e da região situada entre a nervura e o bordo do limbo (% epiderme das faces adaxial e abaxial e espessura da folha). Os dados das variáveis quantitativas foram submetidos aos testes estatísticos multivariados de Análise de Agrupamento e Análise de Componentes Principais. Houve formação de três grupos principais: o primeiro foi constituído pelos três acessos de E. najas; o segundo, por quatro acessos de E. densa; e o terceiro, por apenas um acesso de E. densa. O caráter que mais contribuiu para a diferenciação entre os acessos foi a % feixe vascular da nervura central, seguido da % epiderme da face abaxial da nervura central e % epiderme das faces adaxial e abaxial da região entre a nervura e o bordo foliar. Concluiu-se que a utilização de caracteres anatômicos quantitativos permitiu auxiliar na diferenciação dos acessos e das espécies estudadas; entretanto, devem ser incrementados os estudos relacionando a estrutura anatômica com a resistência e suscetibilidade aos herbicidas.