Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease-associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd-GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd-Brazil), and indicated hybridization between Bd-GPL and Bd-Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400-km transect of the Atlantic Forest. Bd-Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd-GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd-GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd-GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.

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Abbreviations: aerobactin (iucD), afimbrial adhesin (afaB/C), cytolethal distending toxin (cdtB), cytotoxic necrotizing factor type 1 (cnf-1), G adhesin classes of P fimbriae (papG alleles), group II capsule (kpsMTII), α-hemolysin (hly), minor structural subunits of P fimbriae (papE/F), outer membrane protein of P fimbrae (papC), polymerase chain reaction (PCR), S fimbriae (sfaC/D), type 1 fimbriae (fimH), urinary tract infection (UTI), uropathogenic specific protein (usp), uropathogenic Escherichia coli (UPEC), virulence factors (VFs SUMMARYAdhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE /F, papG alleles, fimH, sfa/foc, afaE, hly, usp, cdtB, iucD, and kpsMTII, all

Two hundred and five Escherichia coli strains isolated from calves with diarrhea from mid-western Brazil were screened for the presence of virulence factors associated with bovine colibacillosis. One hundred and two (49.8%) of the E. coli strains produced toxins: Shiga toxins 1 (9.7%) and 2 (6.3%), α-hemolysin (9.7%), enterohemolysin (6.8%), Cytotoxic Necrotizing Factors type 1 (0.5%), and type 2 (4.4%), enterotoxins LT-II (8.3%) and STa (3.9%). No strain produced enterotoxin LT-I. Fimbrial adhesins F5 and F17 were produced by 7.3% and 4.8% of the strains, respectivly, and none expressed F41. Seven strains (3.4%) possessed the gene eae and belonged to serotypes O26:H-; O111:H-and O118:H16. These results suggest that calves in Brazil may be an important source of pathogenic E. coli for animals and humans.

Sex ratio distortion induced by a male-killing agent has been found to a ect Drosophila melanogaster. The trait was discovered accidentally in a collection of¯ies from markets in Campinas, SaÄ o Paulo State, Brazil. Repeated crosses with Canton-S males (for 15 generations to date) and successful transmission using the injection of macerates of sex ratio¯ies, have shown that the trait is inherited maternally, is cytoplasmic and is infectious. Crosses with strains marked with the visible mutation white and viability experiments at pre-adult stages of development, indicate that the skewed sex ratio results from male mortality before hatching. Males do not transmit the trait to their progeny.

Habitat fragmentation and wildlife disease are two widespread drivers of biodiversity loss, yet few empirical studies have explored their interactions. In this study, we utilized a naturally fragmented island system to examine the impacts of fragmentation on genetic diversity and amphibian infection dynamics. We determined the impacts of fragmentation on genetic diversity at the immunity locus MHC IIB, a hypothesized predictor of disease susceptibility. Contrary to the expectation that MHC diversity would remain high due to balancing selection, island populations lost genetic diversity at this locus while simultaneously experiencing positive selection at MHC IIB. We then used Next-Generation Sequencing to identify a variety of potential eukaryotic parasites from amphibian skin swabs. Island populations exhibited higher potential parasite richness (proportion of eukaryotic microbe operational taxonomic units or OTUs from parasitic taxa) relative to mainland populations. MHC homozygotes hosted a lower diversity of potential parasites, and population-level MHC diversity was negatively associated with parasite richness. Our results show that genetic erosion can occur at the MHC IIB locus following fragmentation, which may contribute to increased susceptibility to parasites.

Rozella is a genus of unwalled endoparasites of a variety of hosts including Oomycota (Stramenopiles), Blastocladiomycota and Chytridiomycota (Fungi), and one green alga (Coleochaete, Chlorophyceae). It currently includes more than 20 formally described species, and no new species of Rozella have been described since 1987. We discovered a new Rozella species parasitizing Rhizoclosmatium globosum (Chytridiales, Chytridiomycota) and investigated its morphology, ultrastructure, and phylogenetic position. Herein named as Rozella rhizoclosmatii sp. nov., the organism induces hypertrophy of the host. Its zoospore is ultrastructurally similar to that of Rozella allomycis, although it has a unique zoospore ultrastructural feature, a lattice of perpendicular rods about the nucleus. The 18S rDNA molecular sequence of R. rhizoclosmatii is similar to that of the previously sequenced 'Rozella ex Rhizoclosmatium'. This is the first study to inclusively characterize a new species of Rozella with morphological, ultrastructural and molecular data. As this is only the second Rozella species to be examined ultrastructurally, and because it is parasitic on a member of Chytridiomycota and not Blastocladiomycota, this research supports the conservative nature of zoospore ultrastructure to help define the genus.