Characterization of Hepatozoon spp. in Leptodactylus chaquensis and Leptodactylus podicipinus from two regions of the Pantanal, state of Mato Grosso do Sul, Brazil.

Hepatozoon sp. are parasites that commonly infect frogs and arthropod vectors. This species has variability in the morphological and morphometric characteristics. Due to these variations, the naming of the species is thus impaired and only by visualizing the sporogonic cycle in vector and by molecular studies this problem can be solved. Recently, the use of molecular genetics has helped the species denomination. In this work, we collected 145 frogs (68 Leptodactylus chaquensis and 77 Leptodactylus podicipinus) in different sampling sites, where were found 18 (26.47%) L. chaquensis and 24 (31.17%) L. podicipinus parasitized; besides of gamonts, schizogonic forms were also seen in animals organs. The positivity difference between the collection sites for both frog species was not significant (p = 0.958). Comparing gamonts found in each species of anuran, we observed differences in morphology. The comparison in the molecular level for L. podicipinus was not possible due to small amount of blood obtained, just L. chaquensis had their parasites DNA sequenced. The amplified and sequenced samples, named HEP1 to HEP10, are presented in the phylogenetic tree as a different branch from other haemogregarines described on other hosts. Therefore, we have seen that, although the morphology and morphometry of the collected parasites at each site showed differences, the sequencing of these samples revealed identical species of Hepatozoon, and different compared to those from GenBank, thereby demonstrating that the species of Hepatozoon in L. chaquensis observed in this study probably represent a new species.

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters.

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20°C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America.

Mitochondrial DNA polymorphism and heteroplasmy in populations of Aedes aegypti in Brazil.

The tropical mosquito Aedes aegypti (Diptera: Culicidae) is the most important domestic vector of urban yellow fever and dengue viruses. Ae. aegypti originated from Africa and was probably introduced into Brazil during the colonial period through embarkations, and dengue epidemics soon followed. Genetic analysis of 12 Ae. aegypti populations from five states in Brazil was conducted based on two mitochondrial DNA fragments: cytochrome oxidase I and NADH dehydrogenase subunit 4. Analyses comparing individual haplotypes indicated the existence of two well-defined clades, probably representing two mitochondrial lineages. Analysis of molecular variance showed significant variability in genetic structure among collections within groups. Mantel regression analysis showed a correlation between genetic and geographic distances, mainly because of northern and northeastern populations, in comparison with those in the southeast. The population from Santos, the largest port in Brazil, showed the greatest diversity, with 10 unique haplotypes, an indication of recent introductions that have not yet spread to other Brazilian cities. Different mitochondrial DNA sequences were found in three specimens, indicating the presence of heteroplasmy.

First detection of feline hemoplasmas in free-ranging jaguars (Panthera onca).

Species of hemoplasmas have been described worldwide, but little information is available for wild felids. Between February 2000 and January 2010, blood samples were collected from 30 jaguars (Panthera onca) and 22 domestic cats (Felis catus) from the Cerrado, Pantanal and Amazon biomes of Brazil. In all samples molecular tests were performed for Mycoplasma haemofelis/Mycoplasma haemocanis (Mhf/Mhc), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis' (CMt). Twenty-two (73.4%) jaguars and four domestic cats (18.2%) tested positive for infection with at least one feline hemoplasma: 73.4% jaguars from the three areas were positive for CMhm, 13.6% jaguars from the Pantanal and 50.0% from the Amazon were positive for Mhf/Mhc, and 9.1% of individuals from the Pantanal tested positive for CMt. Domestic cats from the Cerrado (28.6%) and the Pantanal (30.0%) were positive for feline hemoplasma. All but one jaguar from the three sites are healthy. One female adult jaguar showed low body weight and dehydration. This is the first record of feline hemoplasmas in free-ranging jaguars. The high prevalence of CMhm suggest the participation of jaguars in the maintenance of this hemoplasma in nature. Although susceptible to Mhf/Mhc and CMt, jaguars did not appear to participate in the maintenance of these agents in the environment. The involvement of domestic cats in the transmission of any of these hemoplasmas cannot be excluded.

Hepatozoon SPP. Infect Free-Ranging Jaguars (Panthera onca) in Brazil.

This study investigated the presence of Hepatozoon spp. in jaguars ( Panthera onca ) and domestic animals in the Cerrado, Amazon, and Pantanal biomes of Brazil. Between February 2000 and January 2010, blood samples were collected from 30 jaguars, 129 domestic dogs ( Canis lupus familiaris), and 22 domestic cats ( Felis catus ) for molecular tests. All of the jaguars from the Pantanal (n = 22) and Cerrado (n = 4) and 3 of 4 jaguars from the Amazon were positive for Hepatozoon spp. Domestic dogs (62.8%) and cats (31.8%) were also positive for the agent. Hepatozoon nucleotide sequences from jaguars and domestic cats grouped with other Hepatozoon felis, whereas Hepatozoon from domestic dogs showed high similarity to Hepatozoon canis. Different species of Amblyomma were identified as parasitizing the jaguars and may act as vectors for Hepatozoon spp. Jaguars from the 3 sites were healthy and did not seem to be threatened by the hemoparasite within its population or environments. Most likely, jaguars play an important role in the maintenance of Hepatozoon spp. in nature.

Antimicrobial susceptibility of Streptococcus suis isolated from clinically healthy swine in Brazil.

Streptococcus suis is an important pathogen in the swine industry. This study is the first to report on the antimicrobial susceptibility of S. suis isolated from clinically healthy pigs in Brazil; the fourth major pork producer in the world. The antimicrobial susceptibility of 260 strains was determined by disc diffusion method. Strains were commonly susceptible to ceftiofur, cephalexin, chloramphenicol, and florfenicol, with more than 80% of the strains being susceptible to these antimicrobials. A high frequency of resistance to some of the antimicrobial agents was demonstrated, with resistance being most common to sulfa-trimethoprim (100%), tetracycline (97.69%), clindamycin (84.61%), norfloxacin (76.92%), and ciprofloxacin (61.15%). A high percentage of multidrug resistant strains (99.61%) were also found. The results of this study indicate that ceftiofur, cephalexin, and florfenicol are the antimicrobials of choice for empirical control of the infections caused by S. suis.

Streptococcus suis est un pathogène important de l'industrie porcine. Cette étude est la première à rapporter la susceptibilité antimicrobienne de souches S. suis isolées de porcs cliniquement sains provenant du Brésil, le quatrième producteur de porc à l'échelle de la planète. La susceptibilité antimicrobienne de 260 souches fut examinée par la méthode de diffusion des disques imprégnés d'antibiotiques. Plusieurs souches étaient susceptibles au ceftiofur, céphalexine, chlorphénicol, et florfénicol, avec plus de 80 % des souches qui étaient susceptibles à ces antimicrobiens. Une haute fréquence de résistance à certains des antimicrobiens futs démontrée, principalement au sulfa-triméthoprime (100 %), tetracycline (97,69 %), clindamycine (84,61 %), norfloxacine (76,92 %), et ciprofloxacine (61,15 %). Un pourcentage élevé de souches multirésistantes (99,61 %) a également été observé. Les résultats de cette étude indiquent que le ceftiofur, la céphalexine et le florfénicol sont les antimicrobiens de choix pour le contrôle empirique des infections causées par S. suis.(Traduit par les auteurs).

Investigation of tick vectors of Hepatozoon canis in Brazil.

Hepatozoon canis is a common apicomplexan parasite of dogs. In Brazil, in addition to Rhipicephalus sanguineus, Amblyomma ovale, Amblyomma cajennense, and Rhipicephalus (Boophilus) microplus have been suggested to act as vectors. The present study aimed to evaluate, under controlled conditions, the acquisition of H. canis by A. ovale, R. sanguineus, and A. cajennense after feeding on naturally infected dogs. Cytological and histophatological examinations were performed to recover oocysts and other sporogonic stages of the protozoan from the experimentally infected nymphs and adults. None of the R. sanguineus (n=30) or A. cajennense nymphs (n=15) that were dissected after feeding on H. canis naturally infected dogs became infected by the hemoparasite. Likewise, none of the R. sanguineus (n=165) and A. cajennense (n=114) adult ticks that were fed as nymphs on dogs demonstrated infection. Additionally, A. cajennense adult ticks were incapable of acquiring the infection, since no parasite was found in 62 adults that fed on H. canis-infected dogs. With regard to A. ovale ticks, 2 different infestations were carried out. Firstly, a dog with naturally occurring hepatozoonosis was infested with A. ovale adults originating from Rondônia, Brazil. Ticks fed to full engorgement. A total of 31 adults was collected from the dog and dissected on the third day after natural detachment. Oocysts were detected in 13 (42%) of the ticks. The second experimental infestation was carried out using adult ticks originating from São Paulo, Brazil. Surprisingly, of the 103 dissected ticks, only one (1%) contained oocysts in the hemocoel. No other sporogonic stage was found. Results indicate that different strains of A. ovale ticks may exist in Brazil with different susceptibilities to pathogens. Furthermore, it is possible that R. sanguineus and A. cajennense have little or no importance in the transmission of H. canis in rural areas of Brazil.

Seasonal population dynamics and the genetic structure of the mosquito vector Aedes aegypti in São Paulo, Brazil.

Population genetic studies of insect vectors can generate knowledge to improve epidemiological studies focused on the decrease of pathogen transmission. In this study, we used nine SNPs across the Aedes aegypti genome to characterize seasonal population variations of this important dengue vector. Mosquito samples were obtained by ovitraps placed over Botucatu SP from 2005 to 2010. Our data show that, regardless of the large variation in mosquito abundance (deduced from the number of eggs obtained from ovitraps), the effective population size remained stable over the years. These results suggest that Ae. aegypti is able to maintain a sufficiently large active breeding population during the dry season to keep genetic frequencies stable. These results open new perspectives on mosquito survey and control methods.

Genus paracoccidioides: Species recognition and biogeographic aspects.

BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. METHODOLOGY/PRINCIPAL FINDINGS: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11-32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. CONCLUSIONS/SIGNIFICANCE: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides.

Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil.

The status of Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil was studied. For this, 150 animals were examined by blood smears and by PCR; the presence of tick infestation was also investigated. By the blood smear examination, 3 animals (2%) were detected positive and by PCR for Babesia spp. 12 (8%) were positive, with bands visualized in 450 bp. Rhipicephalus sanguineus or Amblyomma spp. were found on 36 (24%) of the 150 dogs. Amblyomma species found were A. cajennense (9/36-25%) and A. ovale (9/36-25%). It was not possible to correlate the presence of R. sanguineus and the infection with Babesia spp. The sequencing of four positive samples demonstrated close identity with B. canis vogeli already characterized in Brazil.

Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil / Infecção por Babesia spp. em cães de áreas rurais do estado de São Paulo, Brasil

The status of Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil was studied. For this, 150 animals were examined by blood smears and by PCR; the presence of tick infestation was also investigated. By the blood smear examination, 3 animals (2 percent) were detected positive and by PCR for Babesia spp. 12 (8 percent) were positive, with bands visualized in 450 bp. Rhipicephalus sanguineus or Amblyomma spp. were found on 36 (24 percent) of the 150 dogs. Amblyomma species found were A. cajennense (9/36-25 percent) and A. ovale (9/36-25 percent). It was not possible to correlate the presence of R. sanguineus and the infection with Babesia spp. The sequencing of four positive samples demonstrated close identity with B. canis vogeli already characterized in Brazil.

A presença de infecção por Babesia spp. em cães de áreas rurais do Estado de São Paulo, Brasil foi investigada. Para tanto, 150 cães foram examinados por técnicas parasitológicas de esfregaços sanguíneos e moleculares (PCR), e também, foi verificada a presença de carrapatos nestes animais. Pela análise de esfregaços sanguíneos, 3 (2 por cento) dos cães estavam infectados, enquanto pela PCR, 12 (8 por cento) dos animais foram positivos com bandas aproximadas de 450 pares de base (pb). Foram observados 36 (24 por cento) cães infestados com Rhipicephalus sanguineus ou com Amblyomma spp. As espécies de Amblyomma observadas foram A. cajennense (25 por cento) e A. ovale (25 por cento). Não foi possível correlacionar a presença de R. sanguineus com a infecção por Babesia spp. O seqüenciamento de quatro amostras positivas demonstrou alta identidade com B. canis vogeli, já caracterizada no Brasil.

Genetic variability in geographical populations of Aedes aegypti (Diptera, Culicidae) in Brazil elucidated by molecular markers

The tropical mosquito, Aedes aegypti is the most important domestic vector of urban yellow fever and dengue. Genetic population studies on this vector are important because they may lead to new tools for surveillance. An analysis of genetic structure was conducted among populations of A. aegypti from 11 localities in four demographic regions within six Brazilian federal states. Markers included 21 random amplified polymorphic DNA (RAPD) loci. RAPD markers were detected among populations and cluster analysis revealed two main groups. We found high genetic polymorphism (H S = 0.224) and high levels of genetic differentiation between populations from different states (G ST = 0.430), as well as in populations from cities in the same state (G ST = 0.410). These results indicate significant differentiation in A. aegypti populations in Brazil. Regression analyses of geographic distances and pairwise F ST values estimated from RAPD markers showed that there is a correlation between genetic structure and geographic localization.

Human respiratory syncytial virus detection in children admitted at a community hospital in Botucatu, SP, Brazil

O Vírus Respiratório Sincicial Humano (VRSH) é descrito como o mais importante patógeno viral causador de doenças respiratórias agudas das vias respiratórias inferiores em crianças. Neste estudo 84 amostras de crianças com idade abaixo dos dois anos apresentando sintomas de doença respiratória aguda, foram obtidas no período de setembro de 2000 a novembro de 2001. Analise por imunofluorescência indireta e transcrição reversa seguida de PCR, revelou que 18 per center (15/84) das amostras foram positivas, sendo que em 80 per center (12/15) dos casos a detecção de VRSH foi observada em crianças abaixo dos seis meses, e também que os subgrupos A e B co-circularam. Estes são os primeiros dados obtidos para a cidade de Botucatu, sendo que a sazonalidade mostrou-se evidente pela maior circulação desse vírus entre os meses de maio e julho.