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The canine heartworm, Dirofilaria immitis, is among the most important parasites of dogs in the United States and worldwide, and may cause severe and potentially fatal disease. Current diagnostic recommendations rely on serological detection of an adult female antigen, and visualization of microfilariae in the blood. Therefore, a reliable diagnosis can be only performed approximately six months post-infection. There is a growing need to characterize novel diagnostic markers that are capable of detecting the early stages of heartworm infection, in special markers associated with third-stage larvae (L3) and fourth-stage larvae (L4). The early detection of infection would guide medical interventions that could impede the development of patent infections and further parasite transmission. We cultured D. immitis L3 and L4 of two laboratorial strains with different susceptibility statuses to macrocyclic lactone drugs in vitro. Excretory/secretory microRNAs were sequenced and analyzed. We identified two miRNA novel candidates secreted abundantly by both L3 and L4 of both strains. These candidates were previously detected in the secretions of other D. immitis stages and one of them was found in the blood of D. immitis-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be D. immitis-specific diagnostic biomarkers, which could allow for the early detection of infection.

Background Amblyomma cajennense F. is one of the best known and studied ticks in the New World because of its very wide distribution, its economical importance as pest of domestic ungulates, and its association with a variety of animal and human pathogens. Recent observations, however, have challenged the taxonomic status of this tick and indicated that intraspecific cryptic speciation might be occurring. In the present study, we investigate the evolutionary and demographic history of this tick and examine its genetic structure based on the analyses of three mitochondrial (12SrDNA, d-loop, and COII) and one nuclear (ITS2) genes. Because A. cajennense is characterized by a typical trans-Amazonian distribution, lineage divergence dating is also performed to establish whether genetic diversity can be linked to dated vicariant events which shaped the topology of the Neotropics. Results Total evidence analyses of the concatenated mtDNA and nuclear + mtDNA datasets resulted in well-resolved and fully congruent reconstructions of the relationships within A. cajennense . The phylogenetic analyses consistently found A. cajennense to be monophyletic and to be separated into six genetic units defined by mutually exclusive haplotype compositions and habitat associations. Also, genetic divergence values showed that these lineages are as distinct from each other as recognized separate species of the same genus. The six clades are deeply split and node dating indicates that they started diverging in the middle-late Miocene. Conclusions Behavioral differences and the results of laboratory cross-breeding experiments had already indicated that A. cajennense might be a complex of distinct taxonomic units. The combined and congruent mitochondrial and nuclear genetic evidence from this study reveals that A. cajennense is an assembly of six distinct species which have evolved separately from each other since at least 13.2 million years ago (Mya) in the earliest and 3.3 Mya in the latest lineages. The temporal and spatial diversification modes of the six lineages overlap the phylogeographical history of other organisms with similar extant trans-Amazonian distributions and are consistent with the present prevailing hypothesis that Neotropical diversity often finds its origins in the Miocene, after the Andean uplift changed the topology and consequently the climate and ecology of the Neotropics.

An investigation of ×19 soil samples collected under the auspices of the Australian citizen science initiative, Soils for Science, returned ×559 chemically dereplicated microbial isolates, of which ×54 exhibited noteworthy anthelmintic activity against either the heartworm Dirofilaria immitis microfilaria and/or the gastrointestinal parasite Haemonchus contortus L1–L3 larvae. Chemical (GNPS and UPLC-DAD) and cultivation (MATRIX) profiling prompted a detailed chemical investigation of Streptomyces sp. S4S-00196A10, which yielded new anthelmintic polyketide goondapyrones A–J (1–10), together with the known actinopyrones A (11) and C (12). Structures for 1–12 were assigned on the basis of detailed spectroscopic and chemical analysis, with preliminary structure activity relationship analysis revealing selected γ-pyrones >50-fold and >13-fold more potent than isomeric α-pyrones against D. immitis mf motility (e.g., EC50 0.05 μM for 1; EC50 2.7 μM for 5) and H. contortus L1–L3 larvae development (e.g., EC50 0.58 μM for 1; EC50 8.2 μM for 5), respectively.