Genotyping of Brazilian Erysipelothrix spp. strains by amplified fragment length polymorphism

One hundred and fifty-one Erysipelothrix spp. isolates from diseased and carrier swine from Brazil were identified by PCR, submitted to serotyping and analyzed by amplified fragment length polymorphism with a single enzyme (AFLP). Reference strains from Australia and the United Kingdom were also examined. The 151 strains were classified into 18 different serotypes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 and 25), being serotype 2b the most frequent (39.7%). By associating serotyping and PCR results, it was possible to identify 146 strains as E. rhusiopathiae and five strains as E. tonsillarum. Despite the fact that for this genus AFLP did not cluster all isolates according to serotype, origin, disease or isolation data, the execution of the technique was easy and fast, demonstrating high discriminatory power. The results produced by the AFLP analysis of Erysipelothrix spp. could also support its use as a discriminatory tool for E. rhusiopathiae and E. tonsillarum species. (C) 2010 Elsevier B.V. All rights reserved.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil

We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. (C) 2011 Elsevier B.V. All rights reserved.

Genotyping of Cryptosporidium spp. from free-living wild birds from Brazil

In wild and domestic birds, cryptosporidiosis is often associated with infections by Cryptosporidium galli, Cryptosporidium baileyi and Cryptosporidium meleagridis. In addition to these species, a number of avian Cryptosporidium species yet to be fully characterized are commonly found among exotic and wild avian isolates. The present study aimed to detect and identify samples of Cryptosporidium spp. from free-living wild birds, in order to contribute to the knowledge of the variability of this parasite in the free-living population of Brazil. Stool samples were collected from 242 birds, with the following proportions of individuals: 50 Emberizidae (20.7%), 112 Psittacidae (46.3%), 44 Cardinalidae (18.2%), 12 Turdidae (5.0%), eight Ramphastidae (3.3%), seven Icteridae (2.9%), three Estrilididae (1.2%), two Contigidae (0.8%), two Thraupidae (0.8%) and two Fringilidae (0.8%). Among the 242 fecal samples from wild birds, 16(6.6%) were positive for the presence of oocysts of Cryptosporidium. Molecular characterization of the 16 samples of Cryptosporidium, were performed with phylogenetic reconstructions employing 292 positions of 18S rDNA. None of the samples of birds was characterized as C meleagridis. C gall was identified in one rufous-bellied thrush (Turdus rufiventris), five green-winged saltators (Saltator similis), one slate-coloured seedeater (Sporophila schistacea), one goldfinch (Carduelis carduelis) and three saffron finches (Sicalis flaveola). One goldfinch isolate, one buff-fronted seedeater (Sporophila frontalis), one red-cowled cardinal (Paroaria dominicana) and one other saffron finch (S. flaveola) were identified as C. baileyi. Avian genotype II was found in an isolate from a white-eyed parakeet (Aratinga leucophthalma). Clinical symptoms of cryptosporidiosis in birds have already been described and the number of wild birds which were shedding parasites was high. Therefore, further epidemiological research and disease surveillance of birds in the wild is warranted. (C) 2010 Elsevier B.V. All rights reserved.

Seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region

Community-acquired pneumonia (CAP) is a common cause of morbidity among children. Evidence on seasonality, especially on the frequency of viral and bacterial causative agents is scarce; such information may be useful in an era of changing climate conditions worldwide. To analyze the frequency of distinct infections, meteorological indicators and seasons in children hospitalized for CAP in Salvador, Brazil, nasopharyngeal aspirate and blood were collected from 184 patients aged < 5 y over a 21-month period. Fourteen microbes were investigated and 144 (78%) cases had the aetiology established. Significant differences were found in air temperature between spring and summer (p = 0.02) or winter (p < 0.001), summer and fall (p = 0.007) or winter (p < 0.001), fall and winter (p = 0.002), and on precipitation between spring and fall (p = 0.01). Correlations were found between: overall viral infections and relative humidity (p = 0.006; r = 0.6) or precipitation (p = 0.03; r = 0.5), parainfluenza and precipitation (p = 0.02; r = -0.5), respiratory syncytial virus (RSV) and air temperature (p = 0.048; r = -0.4) or precipitation (p = 0.045; r = 0.4), adenovirus and precipitation (p = 0.02; r = 0.5), pneumococcus and air temperature (p = 0.04; r = -0.4), and Chlamydia trachomatis and relative humidity (p = 0.02; r = -0.5). The frequency of parainfluenza infection was highest during spring (32.1%; p = 0.005) and that of RSV infection was highest in the fall (36.4%; p < 0.001). Correlations at regular strength were found between several microbes and meteorological indicators. Parainfluenza and RSV presented marked seasonal patterns.

The role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country

We report an investigation for 16 bacteria and viruses among 184 children hospitalized with pneumonia in Salvador, Brazil. Etiology was established in 144 (78%) cases. Viral, bacterial, and mixed infections were found in 110 (60%), 77 (42%), and 52 (28%) patients, respectively. Rhinovirus (21%) and Streptococcus pneumoniae (21%) were the most common pathogens. Our results demonstrate the importance of viral and pneumococcal infections among those patients.

Intratemporal facial nerve ultrastructure in patients with idiopathic facial paralysis. Viral infection evidence study

The etiology of idiopathic peripheral facial palsy (IPFP) is still uncertain; however, some authors suggest the possibility of a viral infection. Aim: to analyze the ultrastructure of the facial nerve seeking viral evidences that might provide etiological data. Material and Methods: We studied 20 patients with peripheral facial palsy (PFP), with moderate to severe FP, of both genders, between 18-60 years of age, from the Clinic of Facial Nerve Disorders. The patients were broken down into two groups - Study: eleven patients with IPFP and Control: nine patients with trauma or tumor-related PFP. The fragments were obtained from the facial nerve sheath or from fragments of its stumps - which would be discarded or sent to pathology exam during the facial nerve repair surgery. The removed tissue was fixed in 2% glutaraldehyde, and studied under Electronic Transmission Microscopy. Results: In the study group we observed an intense repair cellular activity by increased collagen fibers, fibroblasts containing developed organelles, free of viral particles. In the control group this repair activity was not evident, but no viral particles were observed. Conclusion: There were no viral particles, and there were evidences of intense activity of repair or viral infection.

Influence of Viral Infection on Essential Oil Composition of Ocimum basilicum (Lamiaceae)

Ocimum basilicum L., popularly known as sweet basil, is a Lamiaceae species whose essential oil is mainly composed of monoterpenes, sesquiterpenes and phenylpropanoids. The contents of these compounds can be affected by abiotic and biotic factors such as infections caused by viruses. The main goal of this research was an investigation of the effects of viral infection on the essential oil profile of common basil. Seeds of O. basilicum L. cv. Genovese were sowed and kept in a greenhouse. Plants presenting two pairs of leaves above the cotyledons were inoculated with an unidentified virus isolated from a field plant showing chlorotic yellow spots and foliar deformation. Essential oils of healthy and infected plants were extracted by hydrodistillation and analyzed by GCMS. Changes in essential oil composition due to viral infection were observed. Methyleugenol and p-cresol,2,6-di-tert-butyl were the main constituents. However, methyleugenol contents were significantly decreased in infected plants.

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-la Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-la NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model. (C) 2011 Elsevier Ltd. All rights reserved.

Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding

Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rDNA locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi. T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. (C) 2010 Elsevier B.V. All rights reserved.

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp isolated from tap water in Brazil

Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (similar to 99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 degrees C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2 mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01. (C) 2009 Elsevier Inc. All rights reserved.

A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, Were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TO-tat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded loss parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype wall found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

Trypanosoma rangeli isolates of bats from Central Brazil: Genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T rangeli,.T. dionisii, T cruzimarinkellei and T cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus plamirostris were identified as T rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T rangeli and T cruzi. (c) 2008 Elsevier B.V. All rights reserved.