Canine nematode and Giardia spp. infections in dogs in Edmonton, Alberta, the "CANIDA" study.

BACKGROUND: Canine intestinal parasite prevalence may be influenced by geographical region, age, and health status of the dog. Behaviors such as predation, scavenging, or roaming as well as routine administration of anthelmintics also play a role. The purpose of this study was to evaluate fecal test results using zinc sulfate flotation by centrifugation combined with coproantigen testing directed at protein antigens excreted or secreted by hookworms (Ancylostoma spp. Uncinaria stenocephala), ascarids (Toxocara canis, Toxascaris spp. Baylisascaris spp.), whipworms (Trichuris vulpis), and Giardia spp. during active infection in owned dogs visiting dog parks in Western Canada. METHODS: A total of 774 participants were recruited from Edmonton, Alberta, Canada. Canine fecal samples were collected from seven dedicated off-leash dog parks. Participating dog owners responded to a questionnaire regarding their dogs' signalment, previous veterinary history, and use of parasite-preventive products. Fecal samples were tested using zinc sulfate centrifugation combined with coproantigen testing. RESULTS: The overall prevalence of canine intestinal parasites in client-owned dogs was similar to previous studies conducted in the US. Mean age of dogs tested was 4 years, with puppies and older dogs having higher rates of infection than the mean. Fecal flotation centrifugation found 3.2% hookworm, ascarid, whipworm, and Giardia spp.-positive infections. Coproantigen testing identified 5.8% positive infections, including all of the above that were detected using fecal flotation centrifugation. CONCLUSIONS: Coproantigen testing detected more hookworm, ascarid, whipworm, and Giardia spp.-positive samples in addition to detecting all positive results found using fecal flotation centrifugation. Fecal flotation centrifugation combined with coproantigen testing improves sensitivity over flotation alone and may detect pre-patent or sub-clinical infections in dogs visiting public dog parks.

Assessing the potential for infections of Echinococcus multilocularis in dogs in a hotspot of human alveolar echinococcosis infections in North America.

Echinococcus multilocularis is a zoonotic tapeworm, whose metacestode larval stage is the etiological agent for alveolar echinococcosis in humans and is a parasite of emerging concern according to the World Health Organization which is difficult to diagnose and has a case mortality rate of >90% when left untreated. Echinococcus multilocularis requires two mammalian hosts to complete its lifecycle: wild and domestic canids as definitive hosts, and small mammals (mostly rodents) as intermediate ones. Because of their close relations with humans, domestic dogs have been indicated as a mean of infection to people. Human alveolar echinococcosis has historically been rare in North America, however, since 2013, at least seventeen diagnoses have been confirmed in Alberta, Canada. Because of this unprecedented series of cases, assessing the frequency of infections in dogs in Alberta is key to estimate risk for dog owners and animal health professionals. This study was carried out in Edmonton to determine the frequency of E. multilocularis infection in domestic dogs and potential risk factors. Fecal samples and corresponding behavior risk surveys were collected from 775 dogs in seven urban off-leash parks within Edmonton city limits during the summer of 2020. A quantitative PCR fecal test was used to diagnose E. multilocularis infection. We found a single case of E. multilocularis infection (1/775) and determined that the overall true prevalence was 0.2% (95% CrI: 0.0-0.7%) corrected for detection sensitivity and specificity. Overall, these findings confirm the presence of E. multilocularis infection in domestic dogs in Edmonton although further work is required to fully understand the risk factors that may contribute to infection and potential transmission to humans.

Synthesis and 12-helical secondary structure of beta-peptides containing (2R,3R)-aminoproline.

[structure: see text] (2R,3R)-Aminoproline, a pyrrolidine-based beta-amino acid, was synthesized and incorporated into hexa-beta-peptide 4. This residue confers water solubility when the ring nitrogen is protonated and allows for 12-helix formation in aqueous solution. Circular dichroism spectra display the 12-helical signature, and 12-helical structure was confirmed by 2D NMR analysis.

Use of parallel synthesis to probe structure-activity relationships among 12-helical beta-peptides: evidence of a limit on antimicrobial activity.

We report structure-activity trends among helix-forming beta-amino acid oligomers that are intended to mimic alpha-helical host-defense peptides. Parallel synthesis of two small, focused beta-peptide libraries allowed us to identify relatively short (11-residue) beta-peptides that display antimicrobial activity. These beta-peptides exhibit selectivity for bacteria relative to human red blood cells. A large hydrophobic helical surface is necessary for antimicrobial activity. Longer analogues (16 residues) of the most active library members were prepared and evaluated. Some of these longer beta-peptides showed very good antimicrobial activity, but none was more active than a previously reported beta-peptide [Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565]. The extensive literature on alpha-helical host-defense peptides and related alpha-peptides indicates that such molecules are seldom active at concentrations below 1 microg/mL, and our results suggest that amphiphilic helical beta-peptides are subject to a comparable limit.

Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides.

We have designed beta-amino acid oligomers that are helical, cationic, and amphiphilic with the intention of mimicking the biological activity of amphiphilic, cationic alpha-helical antimicrobial peptides found in nature (e.g., magainins). We have previously identified a 17-residue beta-peptide (called beta-17) with antibiotic activity similar to that of a magainin derivative against four bacterial species, including two clinical isolates that are resistant to common antibiotics. This beta-peptide displays very low hemolytic activity against human red blood cells, which indicates selectivity for bacterial cells over mammalian cells. Here we examine some of the factors important for activity in this class of beta-peptides. An amphiphilic helix is necessary, because a nonamphiphilic isomer proved to be inactive. The ratio of cationic to hydrophobic residues is also important. Active beta-peptides induce the leakage of beta-galactosidase from treated Bacillus subtilis cells, as do alpha-helical antibiotic peptides, and this similarity suggests that the beta-peptide mode of action involves disruption of microbial membranes. This class of beta-peptides is not degraded by proteases, which bodes well for biological applications.

Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency.

Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.

Interactions of the antimicrobial beta-peptide beta-17 with phospholipid vesicles differ from membrane interactions of magainins.

We have studied the interaction of beta-17, a potent synthetic antimicrobial beta-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, beta-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of membrane interaction relative to magainin II, but both leakage and membrane binding show that beta-17, like magainin II, has strong affinity for membranes containing anionic lipids. This is likely to be an important factor contributing to the antimicrobial specificity of the beta-peptide.

Tolerance of acyclic residues in the beta-peptide 12-helix: access to diverse side-chain arrays for biological applications.

Oligomeric backbones with well-defined conformational propensities can serve as scaffolds for displaying sets of functional groups in specific three-dimensional arrangements. beta-Peptides are particularly interesting in this regard because several distinct secondary structures can be induced by appropriate choice of beta-amino acid substitution pattern.3 The beta-peptide 12-helix (defined by 12-membered ring C=O(i)- -H-N(i + 3) hydrogen bonds) is of particular interest because this helix resembles the alpha-helix. To date 12-helices have been observed in beta-peptides comprised exclusively of residues containing a five-membered ring constraint. Here we show that 12-helical propensity is maintained when some cyclic beta-amino acid residues are replaced with more flexible acyclic residues. This result is important because use of acyclic residues greatly facilitates introduction of diverse side chains at specific sites along the 12-helix. We demonstrate the utility of this advance in the context of antibiotic design.