Parasites reveal movement of bats between the New and Old Worlds

The global distribution of bat taxa indicates that the Atlantic and Pacific Oceans are effective barriers to movement between the Old and New Worlds. For instance, one of the major suborders, Yinpterochiroptera, has an exclusively Old World distribution, and within the other, Yangochiroptera, no species and only five genera are common to both. However, as bats are sometimes blown out to sea, and have colonised isolated islands, occasional natural movement between the New and Old Worlds does appear to be possible. Here we identify new genotypes of a blood parasite, Trypanosoma dionisii, in Old World bats that are closely related to South American strains. Using highly conservative calibration points, divergence of Old and New World strains is estimated to have occurred 3.2-5.0 million years ago (MYA), depending on the method used (upper 95% CL for maximum time 11.4 MYA). The true date of divergence is likely to be considerably more recent. These results demonstrate that taxon-specific parasites can indicate historical movements of their hosts, even where their hosts may have left no lasting phylogenetic footprint. (C) 2012 Elsevier Inc. All rights reserved.

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

Abstract Background Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli. Methods Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy. Results New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. Conclusion Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

BACKGROUND: Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli. METHODS: Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy. RESULTS: New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. CONCLUSION: Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

Molecular characterization by amplified fragment length polymorphism and aflatoxin production of Aspergillus flavus isolated from freshly harvested peanut in Brazil

Brazil contributes substantially to the global peanut production, and the state of Sao Paulo is the largest producer in the country. Peanut crops can be contaminated by Aspergillus flavus strains producing aflatoxins, which are highly toxic and carcinogenic. Thus, the production of high-quality peanuts is crucial both for the commercial peanut industry and as a matter of public health. In this study, we used amplified fragment length polymorphism analysis (AFLP) to investigate the genetic variability among A. flavus strains isolated from fresh peanuts harvested in four different regions in the state of Sao Paulo, and to determine whether the molecular genetic profiles correlated with aflatoxin production or sclerotia formation. AFLP analysis generated 78 fragments ranging from 27 to 365 base pairs in length. Thirteen percent were not polymorphic. Genotyping identified twelve groups of A. flavus. On the basis of the polymorphisms identified, similarity between the isolates ranged from 37% to 100%. Of all isolates collected, 91.7% produced aflatoxins and 83.9% produced small sclerotia. Statistical analysis failed to suggest any relationship between the presence of sclerotia and mean levels of aflatoxins B-1 and B-2. Furthermore, a dendrogram based on AFLP data revealed substantial genetic variability among the A. flavus strains, but showed no correlation between dendrogram groups separated by molecular genetic features and production of aflatoxins B-1 or B-2 or the formation of sclerotia.

cDNA-AFLP analysis of Psidium guajava L. cultivars under water stress and mechanical injury: methodological implications

Studies involving amplified fragment length polymorphism (cDNA-AFLP) have often used polyacrylamide gels with radiolabeled primers in order to establish best primer combinations, to analyze, and to recover transcript-derived fragments. Use of automatic sequencer to establish best primer combinations is convenient, because it saves time, reduces costs and risks of contamination with radioactive material and acrylamide, and allows objective band-matching and more precise evaluation of transcript-derived fragments intensities. This study aimed at examining the gene expression of commercial cultivars of P. guajava subjected to water and mechanical injury stresses, combining analyses by automatic sequencer and fluorescent kits for polyacrylamide gel electrophoresis. Firstly, 64 combinations of EcoRI and MseI primers were tested. Ten combinations with higher number of polymorphic fragments were then selected for transcript-derived fragments recovering and cluster analysis, involving 45 saplings of P. guajava. Two groups were obtained, one composed by the control samplings, and another formed by samplings undergoing stress, with no clear distinction between stress treatments. The results revealed the convenience of using a combination of automatic sequencer and fluorescent kits for polyacrylamide gel electrophoreses to examine gene expression profiles. The Unweighted Pair Group Method with Arithmetic Mean analysis using Euclidean distances points out a similar induced response mechanism of P. guajava undergoing water stress and mechanical injury.

Experimental infection with Neospora caninum in pregnant bitches

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.

Genetic Variants in Matrix Metalloproteinase-9 Gene Modify Metalloproteinase-9 Levels in Black Subjects

Altered matrix metalloproteinases (MMPs) levels are involved in cardiovascular diseases and increased MMP-9 levels enhance the cardiovascular risk in apparently healthy subjects. We investigated the effects of MMP-9 gene polymorphisms and haplotypes on the circulating MMP-9 levels in healthy black subjects and the effects of an MMP-2 polymorphism on the plasma MMP-2 concentrations. We studied 190 healthy subjects, nonsmokers, self-reported as blacks (18-63 years). Genotypes for the MMP-2 C-1306T polymorphism and the MMP-9 C-1562T, 90(CA)(14-24) and Q279R polymorphisms (rs243865, rs3918242, rs2234681, and rs17576, respectively) were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction or restriction fragment length polymorphism. Alleles for the 90(CA)(14-24) polymorphism were grouped as low (L) when there were < 21 and high (H) when there were >= 21 CA repeats. The plasma levels of MMP-2 and MMP-9 were determined by gelatin zymography. The software PHASE 2.1 was used to estimate the haplotypes frequencies. Although we found no effects of the MMP-9 C-1562T or the Q279R polymorphisms on MMP-9 levels, higher MMP-9 levels were associated with the HH genotype for the -90(CA)(14-24) polymorphism compared with the HL or LL genotypes. Lower MMP-9 levels were found in carriers of the CRL haplotype (combining the C, R, and L alleles for the MMP-9 polymorphisms) compared with the CRH haplotype. Consistent with this finding, the CRL haplotype was more commonly found in subjects with low MMP-9 levels. The MMP-2 C-1306T polymorphism had no effects on the plasma MMP-2 levels. Our results show that MMP-9 genetic variations modify MMP-9 levels in black subjects and may offer biochemical evidence implicating MMP-9 in the pathogenesis of cardiovascular diseases in blacks.

Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in Sao Paulo, Brazil

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coil (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coil strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type. (C) 2010 Elsevier Ltd. All rights reserved.

Fc gamma Receptor Gene Polymorphisms in Childhood Immune Thrombocytopenic Purpura

Immune thrombocytopenic purpura (ITP) is a common hematological disorder in the childhood, and it is one of the most common forms of autoimmune disease in pediatric patients. The ITP basis is a primary dysfunction of the immune system. This study aimed to analyze the genetic polymorphisms of the Fc gamma receptors IIA and IIIA. The genetic polymorphisms of the Fc receptors gamma IIA (131H/R) and gamma RIIIA (158V/F) were analyzed by polymerase chain reaction-restriction fragment length polymorphism technique. Odds ratio and 95% confidence interval were calculated by chi(2) test. Homozygous polymorphic genotype for the Fc gamma RIIIA was significantly more frequent among patients compared with controls (odds ratio = 0.27; 95% confidence interval, 0.09-0.80; P = 0.03). There was no statistical difference between the ITP group and the controls in the analysis of combinations of alleles of the high-affinity Fc receptor, but the ITP individuals with this combination had a lower duration of disease (P = 0.01). Genetic polymorphisms in immune system genes can be important for ITP pathogenesis and disease outcome.

Leishmania (Viannia) braziliensis is the main species causing cutaneous leishmaniasis in the Federal District of Brazil

The first autochthonous case of American cutaneous leishmaniasis was reported in the Federal District in 1980, and the species involved in this type of leishmaniasis was unknown. This study aimed to identify the species that causes the disease in the Federal District and to investigate its clinical and epidemiological aspects. Between 2000 and 2007, 71 autochthonous cases of leishmaniasis were reported in the Federal District. Leishmania species were identified by means of direct immunofluorescence reactions using monoclonal antibodies and restriction fragment length polymorphism. The species of 40 (56.33%) out of 71 samples were identified. Thirty-six (90%) were identified as Leishmania (Viannia) braziliensis and four (10%) were identified as Leishmania (Leishmania) amazonensis. In this area, the disease had clinical and epidemiological characteristics similar to those found in other Brazilian regions.

Prevalence of avian pathogenic Escherichia coli (APEC) clone harboring sfa gene in Brazil

Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farmpresented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk.

Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients

Introduction: Vitamin D is responsible for the regulation of certain genes at the transcription level, via interaction with the vitamin D receptor, and influences host immune responses and aspects of bone development, growth, and homeostasis. Our aim was to investigate the association of TaqI vitamin D receptor gene polymorphism with external apical root resorption during orthodontic treatment. Methods: Our subjects were 377 patients with Class II Division 1 malocclusion, divided into 3 groups: (1) 160 with external apical root resorption <= 1.43 mm, (2) 179 with external apical root resorption >1.43 mm), and (3) 38 untreated subjects. External apical root resorption of the maxillary incisors was evaluated on periapical radiographs taken before and after 6 months of treatment. After DNA collection and purification, vitamin D receptor TaqI polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate analyses were performed to verify the association of clinical and genetic variables with external apical root resorption (P <0.05). Results: There was a higher proportion of external apical root resorption in orthodontically treated patients compared with the untreated subjects. In patients orthodontically treated, age higher than 14 years old, initial size of the maxillary incisor root superior to 30 mm, and premolar extraction were associated with increased external apical root resorption. Genotypes containing the C allele were weakly associated with protection against external apical root resorption (CC + CT x TT [odds ratio, 0.29; 95% confidence interval, 0.07-1.23; P = 0.091]) when treated orthodontic patients were compared to untreated individuals. Conclusions: Clinical factors and vitamin D receptor TaqI polymorphism were associated with external apical root resorption in orthodontic patients. (Am J Orthod Dentofacial Orthop 2012; 142: 339-47)

Genetic diversity of Stryphnodendron adstringens (Mart.) Coville determined by AFLP molecular markers

Bark extracts of Stryphnodendron adstringens (Mart) Coville a Leguminosae species, well known in Brazil as barbatimao, are popularly used as healing agent. The objective of this work was to determine the genetic diversity of S. adstringens populations and to correlate genetic distances to the production of tannins. S. adstringens accessions from populations found in Cerrado regions in the states of Goias, Minas Gerais and Sao Paulo were analyzed using the AFLP (Amplified Fragment Length Polymorphism) technique. A total of 236 polymorphic bands were scored and higher proportion of genetic diversity was found inter populations (70.9%), rather than intra populations (29.1%). F-ST value was found to be significantly greater than zero (0.2906), demonstrating the complex genetic structure of S. adstringens populations. Accessions collected in Cristalina, GO, showed higher percentage of polymorphic loci (87.3%) and the highest genetic diversity. The lowest genetic variability was detected among accessions from the population growing in Caldas Novas, GO. The genetic distance among populations was estimated using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which grouped populations into 3 clusters. Moreover, chemotypes with tannin concentration above 40% showed higher genetic similarity. AFLP analysis proved to be an efficient gene mapping technique to determine the genetic diversity among remaining populations of S. adstringens. Obtained results may be employed to implement further strategies for the conservation of this medicinal plant. (C) 2011 Elsevier Ltd. All rights reserved.

Virulence and molecular aspects of Bordetella avium isolated from cockatiel chicks (Nymphicus hollandicus) in Brazil

Bordetella avium is an opportunistic pathogen that presents tropism for ciliated epithelia, leading to upper respiratory tract disease in turkeys. This agent has also been associated with Lockjaw Syndrome in psittacine birds, but literatures describing the importance of this agent in such species are rare. The purpose of the present study was to report the first outbreak of B. avium infection in juvenile cockatiels demonstrating the Lockjaw Syndrome in Brazil and to investigate the antimicrobial resistance profile and phenotypic and genotypic characteristics of these strains. Surprising, the strains obtained from five infected cockatiel chicks from three different breeders from different Brazilian states showed a clonal relationship using the Pulsed Field Gel Electrophoresis and Single Enzyme Amplified Fragment Length Polymorphism techniques. The virulence potentials of the B. avium strains were assessed using tracheal adherence and cytotoxic effects on a VERO cell monolayer. (C) 2012 Elsevier B.V. All rights reserved.