Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo)

Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR), the first specific for species belonging to the Mycobacterium complex (MTBC) and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3%) from 18 animals (60%) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5%), 9 prescapular lymphnodes (33.3%), 2 lungs (7.4%), and 1 liver (3.7%). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Reproductive isolation between sympatric and allopatric Brazilian populations of Lutzomyia longipalpis s.l. (Diptera: Psychodidae)

Lutzomyia longipalpis s.l., the main vector of Leishmania chagasi in Latin America, is a species complex although the exact number of siblings is yet unknown. In Brazil, the siblings differ in male copulatory courtship songs and pheromones that most certainly act as pre-zygotic reproductive barriers. Here we analysed the reproductive isolation between three allopatric and two sympatric populations of Lu. longipalpis s.l. from Brazil. The results indicate a strong copulatory and pre-mating isolation between the three allopatric populations. In addition, the results also indicate a stronger pre-mating isolation between the two sympatric siblings than between the three allopatric ones, suggesting a role for reinforcement in the speciation of the Lu. longipalpis s.l. complex.

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

Lesion aspirate culture for the diagnosis and isolation of Leishmania spp. from patients with cutaneous leishmaniasis

The detection of Leishmania spp. in skin lesion aspirates, using a puncture technique, was evaluated in 76 patients with cutaneous leishmaniasis (CL) who were referred to a Leishmaniasis Reference Centre in Brazil. CL was defined based on skin lesions suggestive of the disease and on a positive result of the Montenegro skin test or Giemsa-stained imprints of biopsy fragments. The aspirates were cultured using a vacuum tube device containing culture medium and evaluated for the presence of Leishmania spp. The biphasic medium culture was examined once a week for three weeks. Promastigotes were observed in 53/76 (69.7%) cultures. Stained smears from 60 of the 76 patients were evaluated using PCR-RFLP to detect the conserved minicircle region of Leishmania spp. and to classify the parasite. Of these patients, 45 (75%) showed positive results in aspirate culture and 15 presented negative results. The PCR was positive in 80% (53/60) samples. The PCR-RFLP profile was determined in 49 samples, of which 45 (92%) showed a pattern compatible with Leishmania (Viannia) braziliensis. The aspirate culture is a sensitive and feasible method for diagnosing CL and may be routinely adopted by health services for L. (V.) braziliensis isolation and identification.

First isolation of Cryptococcus gattii molecular type VGII and Cryptococcus neoformans molecular type VNI from environmental sources in the city of Belém, Pará, Brazil

Cryptococcus neoformans and Cryptococcus gattii are important agents of meningoencephalitis in humans in the city of Belém. This clinical data suggests that the region may be a highly endemic area for the pathogenic Cryptococcus species within the state of Pará (PA), Northern Brazil. Preliminary analysis of 11 environmental samples from the city of Belém showed two positive locations, including a hollow of a kassod tree (Senna siamea) colonized simultaneously by C. gattii molecular type VGII and C. neoformans molecular type VNI, and a birdcage in a commercial aviary positive for C. neoformans, molecular type VNI. This is the first evidence of an environmental occurrence of molecular types VNI and VGII in PA.

Rhodococcus equi isolation from sputum of patients with suspected tuberculosis

Rhodococcus equi has emerged as an opportunistic pathogen associated with pulmonary, invasive or systemic infections in immunocompromised patients. We report the identification of 51 R. equi isolates found in sputum samples of 546 individuals suspected to have pulmonary tuberculosis in two Public Health Hospital Units in Brazil. The epidemiology of R. equi infection as well as the phenotypic identification and drug susceptibility profile of isolates are described in this paper.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae)

We investigated the use of Bacillus thuringiensis isolated in the state of Amazonas, in Brazil, for the biological control of the dengue vector Aedes aegypti. From 25 soil samples collected in nine municipalities, 484 bacterial colonies were obtained, 57 (11.78%) of which were identified as B. thuringiensis. Six isolates, IBt-03, IBt-06, IBt-07, IBt-28, IBt-30, and BtAM-27 showed insecticidal activity, and only BtAM-27 presents the five genes investigated cry4Aa, cry4Ba, cry10Aa, cry11Aa, and cry11Ba. The IBt-07 and IBt- 28, with lower LC50 values, showed equal toxicity compared to the standards. The isolates of B. thuringiensisfrom Amazonas constitute potential new means of biological control for A. aegypti, because of their larvicidal activity and the possibility that they may also contain new combinations of toxins.

Isolation and characterization of two plant growth-promoting bacteria from the rhizoplane of a legume (Lupinus albescens) in sandy soil

Two bacterial strains that amplified part of the nifH gene, RP1p and RP2p, belonging to the genus Enterobacter and Serratia, were isolated from the rhizoplane of Lupinus albescens. These bacteria are Gram-negative, rod-shaped, motile, facultative anaerobic, and fast-growing; the colonies reach diameters of 3-4 mm within 24 h of incubation at 28 ºC. The bacteria were also able to grow at temperatures as high as 40 ºC, in the presence of high (2-3 % w/v) NaCl concentrations and pH 4 -10. Strain RP1p was able to utilize 10 of 14 C sources, while RP2p utilized nine. The isolates produced siderophores and indolic compounds, but none of them was able to solubilize phosphate. Inoculation of L. albescens with RP1p and RP2p strains resulted in a significant increase in plant dry matter, indicating the plant-growth-promoting abilities of these bacteria.

INOCULATION AND ISOLATION OF PLANT GROWTH-PROMOTING BACTERIA IN MAIZE GROWN IN VITÓRIA DA CONQUISTA, BAHIA, BRAZIL

Maize is among the most important crops in the world. This plant species can be colonized by diazotrophic bacteria able to convert atmospheric N into ammonium under natural conditions. This study aimed to investigate the effect of inoculation of the diazotrophic bacterium Herbaspirillum seropedicae (ZAE94) and isolate new strains of plant growth-promoting bacteria in maize grown in Vitória da Conquista, Bahia, Brazil. The study was conducted in a greenhouse at the Experimental Area of the Universidade Estadual do Sudoeste da Bahia. Inoculation was performed with peat substrate, with and without inoculation containing strain ZAE94 of H. seropedicae and four rates of N, in the form of ammonium sulfate (0, 60, 100, and 140 kg ha-1 N). After 45 days, plant height, dry matter accumulation in shoots, percentage of N, and total N (NTotal) were evaluated. The bacteria were isolated from root and shoot fragments of the absolute control; the technique of the most probable number and identification of bacteria were used. The new isolates were physiologically characterized for production of indole acetic acid (IAA) and nitrogenase activity. We obtained 30 isolates from maize plants. Inoculation with strain ZAE94 promoted an increase of 14.3 % in shoot dry mass and of 44.3 % in NTotal when associated with the rate 60 kg ha-1 N. The strains N11 and N13 performed best with regard to IAA production and J06, J08, J10, and N15 stood out in acetylene reduction activity, demonstrating potential for inoculation of maize.

Isolation of a cotton NADP(H) oxidase homologue induced by drought stress

The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L.) genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD) technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H) oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.