Isolation of coccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, São Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-AL, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos an a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

A critical analysis of isolation of Paracoccidioides brasiliensis from soil

The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil. The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al. in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al, in Ibia, Brazil, Ferreira et al, in Uberlandia, Brazil, and Gezuele et al. at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P. brasiliensis. In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis. These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen. The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified.

Isolation of brucella spp from milk of brucellosis positive cows in São Paulo and Minas Gerais states

A brucelose é uma zoonose crônica de importância para a Saúde Pública. Considerando o pequeno número de dados brasileiros sobre a sua presença em leite cru e derivados não-pasteurizados, estudamos a presença de brucelas em leite de animais sorologicamente positivos. A soroaglutinação rápida (SAR), a soroaglutinação lenta (SAL) e a soroaglutinação lenta com tratamento do soro com 2-mercaptoetanol foram utilizados para identificar os animais positivos nas propriedades estudadas. Amostras diárias de 300 ml de leite foram colhidas por três dias de todos os quartos mamários produtivos (75 ml/teto). As amostras eram misturadas e centrifugadas. Parte do sedimento e do sobrenadante foi inoculada em meios de Farrel e Brodie-Sinton (BS) suplementados com agentes antimicrobianos. As placas e tubos foram cultivados por sete dias a 37ºC, em microaerofilia. As colônias suspeitas no meio BS foram imediatamente repicadas para ágar-Brucella, e cultivadas sob a mesma condição. Os microrganismos isolados foram submetidos a procedimentos de identificação, incluindo a coloração de Gram, requerimento de CO2, produção de H2S, atividade da urease e crescimento na presença de tionina e fucsina. Das 49 amostras examinadas, isolou-se Brucella abortus de 15 (30,61%). Os biótipos isolados foram: biótipo 1 em uma amostra (2,04%), biótipo 2 em oito (16,32%) e biótipo 3 em seis amostras (12,25%)

Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue

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

Isolation of Staphylococcus epidermidis from inflamed upper respiratory tract of an orange-spined hairy dwarf porcupine (Sphiggurus villosus)

The orange-spined hairy dwarf porcupine (Sphiggurus villosus) is a rodent species common in most parts of South America, and little is known about the pathologies that can afflict it. A specimen was delivered at the Wildlife Research and Medical Center (CEMPAS), School of Veterinary Medicine and Animal Husbandry, UNESP, Botucatu, SP, Brazil. The animal showed intense apathy, with purulent secretion in the nasal cavity and fracture of the lumbar spine. Due to the unfavorable prognosis, the porcupine was euthanized and microbiological culture of nasal discharge showed Staphylococcus epidermidis. The antimicrobial resistance test revealed sensitivity to all tested antimicrobials (ampicillin, oxacillin, tetracycline, penicillin G, neomycin, cephalexin, gentamicin, enrofloxacin, ciprofloxacin, cotrimoxazol, cefoxitin and cephalothin). This bacterium is part of the nasal flora of humans and other animals, and may cause infection under certain conditions. In the present study, the infection and colonization by S. epidermidis was the probable cause of the inflammatory process. The sensitivity to all tested antimicrobials suggests that this strain has not been previously exposed to such drugs.

Isolation of Salmonella enterica Serovar Enteritidis in Blue-Fronted Amazon Parrot (Amazona aestiva)

Avian salmonellosis is a disease caused by bacteria of the genus Salmonella that can cause three distinct diseases in birds: pullorum diseases, fowl typhoid, and paratyphoid infection. Various wildlife species are susceptible to infections by Salmonella, regardless of whether they live in captivity or freely in the wild. The present study verified the presence of Salmonella enterica serovar Enteritidis in three captive specimens of Amazona aestiva. The study involved a total of 103 birds undergoing rehabilitation to prepare for living in the wild, after having been captured from animal traffickers and delivered to the Centrofauna Project of the Floravida Institute in São Paulo, Brazil. This is the first report of Salmonella Enteritidis isolation in A. aestiva that originated from capture associated with animal trafficking; Salmonella was detected during the study by the serologic method of rapid serum agglutination on a plate with bacterial isolate. The antimicrobial profile exam of the isolated samples demonstrated sensitivity to ampicillin, cefaclor, ciprofloxacin, and cloranfenicol. The three samples also presented resistance to more than four antibiotics. The presence of the genes invA and spvC was verified by PCR technique and was associated with virulence and absence of class 1 integron, a gene related to antimicrobial resistance. The commercial antigen for pullorum disease was shown to be a useful tool for rapid detection in the screening of Salmonella of serogroup D(1) in Psittaciformes. New studies on Salmonella carriage in birds involved in trafficking must be performed to better understand their participation in the epidemiologic cycle of salmonellosis in humans and other animals.

Isolation and multilocus genotyping of Toxoplasma gondii in seronegative rodents in Brazil

Synanthropic rodents, mainly rats and mice, become ecologically associated with men due to changes in their ecosystems caused by human activities. These animals may take part in the epidemiological cycles of several diseases, including toxoplasmosis. The presence of serum antibodies to Toxoplasma gondii in 43 rodents captured in the urban area of Umuarama, PR, Brazil, was verified by modified agglutination test (MAT). Brain and heart samples were also collected and bioassayed in mice for the isolation of the parasite. Isolated samples were analyzed by 12 multilocus genotyping. Although all rodents were seronegative, the parasite was isolated in one mouse (Mus musculus) and one rat (Rattus rattus). Genotyping showed that these samples were similar to those previously isolated from cats in the state of Parana, Brazil. (C) 2010 Elsevier B.V. All rights reserved.

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.

Susceptibility of mammalian cell line for isolation of IBDV from clinical samples

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

Isolation and characterization of microsatellite markers for Cascaria sylvestris Sw. (Salicaceae), a neotropical medicinal tree

Casearia sylvestris Sw. is a widespread neotropical tree utilized in popular medicine. Recent research ranked Casearia as one of the most promising genus in the search of drugs against cancer. Despite its wide distribution and pharmacological importance, no microsatellite markers have yet been developed for this genus. In this study, we provide 10 polymorphic microsatellite loci specifically designed for C. sylvestris, used to analyse 90 individuals distributed in two populations from São Paulo state, Brazil. on average, 12.3 alleles per locus were identified, showing the ability of the markers to detect microsatellite polymorphism in this species.

Isolation and characterization of microsatellite DNA markers for spinner dolphin (Stenella longirostris)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation and characterization of a satellite DNA family in Achirus lineatus (Teleostei : Pleuronectiformes : Achiridae)

Agarose gels stained with Ethidium bromide and Southern blot experiments of HindIII-digested genomic DNA of Achirus lineatus evidenced the presence of monomers and multimers of a DNA segment of about 200 bp, named here Al-HindIII sequence. No signals were observed in Southern blot experiments with genomic DNA of other flatfish species. The DNA sequencing of four recombinant clones showed that Al-HindIII sequences had 204 bp and were 63.72% AT-rich. FISH experiments using a Al-HindIII sequence as probe showed bright signals in the centromeric position of all chromosomes of A. lineatus.

Isolation and characterization of eight microsatellite loci in Leporinus macrocephalus (Characiformes : Anostomidae) and cross-species amplification

Leporinus macrocephalus is a species endemic to the Paraguay River basin and an important fishery resource, as well as a valuable species in aquaculture programs. A total of eight polymorphic microsatellite loci were isolated and characterized. A population survey was conducted involving 45 specimens whereby a large number of alleles (range 5-17 among loci), a highly observed (0.1667-0.6129) and an expected (0.6967-0.9448) heterozygosity was detected, indicating its usefulness in population genetics studies. Cross-species amplification was successful in eight Characiformes species.

Isolation and characterization of microsatellite loci in Pseudoplatystoma corruscans (Siluriformes : Pimelodidae) and cross-species amplification

A total of five polymorphic microsatellites loci from Pseudoplatystoma corruscans were isolated and characterized. A population survey involving 43 specimens resolved a large number of alleles (range seven to eight among loci) and high observed heterozygosity (0.500-0.615), indicating its usefulness in population genetics studies. Cross-species amplification was successful in four other Pimelodidae species.