Explore the vast range of titles available.

The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

Background Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. Methods/principle findings Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. Conclusions The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis.

Background Endemic domestic dog-ruminant cycles and human cystic echinococcosis caused by Echinococcus granulosus have been sporadically reported in the United States. However, there is a paucity of molecular data describing the genotypes and haplotypes of this important cestode in domestic ruminant hosts. Methods Ninety-four cysts from the lungs and/or livers of slaughtered beef cattle (76 samples), dairy cows (five samples) and sheep (13 samples) were collected from abattoirs in four states of the USA. Samples were genotyped at two mitochondrial loci, cox1 and nad5. Sequences were used to determine species, genotypes and haplotypes using median joining networks and Bayesian phylogenetic analyses. Cyst fertility was assessed in hematoxylin and eosin-stained sections. Additionally, previously reported autochthonous E. granulosus infections in the USA in various hosts were mapped. Results Based on cox1 sequences obtained from 94 cysts, 89 (94.7%) were identified as E. granulosus G1/G3, while five (5.3%) were Taenia hydatigena. Taenia hydatigena were only isolated from sheep. Based on nad5 sequences obtained from 89 hydatid cysts, 96.6% and 3.4% belonged to E. granulosus sensu stricto genotypes G1 and G3 respectively. Two haplotypes were found among E. granulosus cox1 sequences, neither of which was geographically unique. Six haplotypes were found among nad5 sequences in genotype G1, of which five were novel, while one haplotype was found in genotype G3. In the concatenated cox1-nad5 dataset, seven haplotypes were identified, of which six were geographically unique. All cysts from cattle were non-fertile. Four cysts from sheep were fertile. Conclusions All genotyped samples belonged to E. granulosus s.s. This is the first study to our knowledge to confirm the presence of genotypes G1 and G3 in domestic cattle and sheep intermediate hosts in the USA and provide data for future diagnostic and epidemiological studies. Sequences have been deposited in GenBank ( cox1 sequences: OR398494-OR398496, nad5 sequences: OR400695-OR400702). Graphical Abstract

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.

Tritrichomonas foetus is a sexually transmitted protozoan that causes early embryonic death in cattle. A challenge in trichomonosis research is that in vivo studies of treatments, diagnostic strategies, and vaccines are severely hampered by the logistical challenge and cost of maintaining adult bulls. Since natural infections are diagnosed in postpubescent animals, the paradigm is that only mature breeding bulls can be infected. In this study, we hypothesized that prepubescent bull calves could be artificially infected with T. foetus trophozoites for the purpose of conducting research trials. Initial attempts to directly infect bull calves with two different parasite isolates resulted in the sporadic and transient detection of parasite DNA but not culturable trophozoites. In vitro and in vivo studies suggested that urine directly inhibited trophozoites, likely by osmotic damage and mechanical flushing action. Studies utilizing a perineal urethrostomy to remove urine flow from the prepuce resulted in the ability to colonize the prepuce, with live organisms being cultured for as long as 15 days post-inoculation. Future studies optimizing this technique have the potential to accelerate the pace of bovine trichomonosis research and may have applications in the study of human trichomoniasis.

Background Deep amplicon sequencing of nematode internal transcribed spacer 2 (ITS2), also referred to as the “nemabiome,” has been increasingly used in veterinary hosts to study gastrointestinal nematodes. While post-sequencing bioinformatic pipelines such as DADA2 and mothur have been optimized, most researchers typically use the DADA2 pipeline in R. For optimal performance, DADA2 needs parameter tuning, which is hard for novices. Methods In this study, we present an implementation of the DADA2 pipeline within QIIME2 for nemabiome analysis and compare its performance against the commonly used R-based DADA2 pipeline. To evaluate performance against samples with known composition, we generated simulated nemabiome datasets representing canine, ruminant, and equine nematode communities. We also tested the pipelines using publicly available datasets from ten veterinary host species. For both pipelines, we evaluated differences in amplified sequence variant (ASV) generation, taxonomic classification, and diversity metrics. We also tested different Idtaxa parameter settings within the R DADA2 pipeline (classification threshold and bootstrap iterations) to understand its effects on nemabiome outcomes. Results While both pipelines showed minor discrepancies in relative abundance estimates, with minimal parameter optimization, QIIME2 outputs were closer to ground truth in simulated datasets. QIIME2 using the scikit Bayes classifier produced fewer unclassified taxa and more consistent species-level identifications compared with R DADA2’s Idtaxa, particularly in complex communities. Community-level differences in beta diversity were primarily driven by differences in taxonomic assignment. Parameter testing revealed that lower classification thresholds in R DADA2 reduced the number of unclassified taxa but increased the risk of misclassification, highlighting the need for careful parameter selection and reporting. Conclusions With minimal parameter tuning, QIIME2 outperformed the R pipeline in taxonomic resolution, and improved reproducibility by provenance tracking. Our findings emphasize how bioinformatics pipeline choices impact nemabiome outputs including the number of species detected, ranks of abundant taxa, and alpha and beta diversities. We provide a reproducible and user-friendly QIIME2 workflow suitable for researchers seeking standardized analyses of ITS2 nemabiome data. Graphical Abstract

Dipylidium caninum (Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host-associated canine and feline genotypes based on infection studies, differences at the 28S rDNA gene, and complete mitochondrial genomes. There have been no comparative genome-wide studies. Here, we sequenced the genomes of a dog and cat isolate of Dipylidium caninum from the United States using the Illumina platform at mean coverage depths of 45× and 26× and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates. Genomes of D. caninum canine and feline genotypes generated in this study, had an average identity of 98% and 89%, respectively, when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein-coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study build a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance.

Abstract Background Canine acaricides with rapid onset and sustained activity can reduce pathogen transmission risk and enhance pet owner experience. This randomized, complete block design, investigator-masked study compared the speed of kill of Amblyomma americanum provided by three monthly-use isoxazoline-containing products. Methods Eight randomized beagles per group were treated (day 0), per label, with sarolaner (combined with moxidectin and pyrantel, Simparica Trio™), afoxolaner (NexGard™), or lotilaner (Credelio™), or remained untreated. Infestations with 50 adult A. americanum were conducted on days − 7, − 2, 21, and 28, and tick counts were performed on day − 5 (for blocking), and at 4, 8, 12, 24, 48, and 72 h following treatment and subsequent infestations. Efficacy calculations were based on geometric mean live tick counts. A linear mixed model was used for between-group comparisons. Results On day 0, only lotilaner significantly reduced an A. americanum infestation by 12 h (43.3%; P  = 0.002). Efficacy of lotilaner and afoxolaner at 24 h post-treatment was 95.3% and 97.6%, respectively, both significantly different from sarolaner (74%) ( P  = 0.002, P  < 0.001, respectively). On day 21, at 12 h postinfestation, lotilaner efficacy (59.6%) was significantly different from sarolaner (0.0%) ( P  < 0.001) and afoxolaner (6.3%) ( P  < 0.001). At 24 h, lotilaner efficacy (97.4%) was significantly different ( P  < 0.001) from sarolaner and afoxolaner (13.6% and 14.9%, respectively). On day 28, at 12 h postinfestation, lotilaner efficacy (47.8%) was significantly different from sarolaner (17.1%) ( P  = 0.020) and afoxolaner (9.0%) ( P  = 0.006). At 24 h, lotilaner efficacy (92.3%) was significantly different from sarolaner 4.9% ( P  < 0.001) and afoxolaner (0.0%) ( P  < 0.001). Speed of kill for sarolaner and afoxolaner, but not lotilaner, significantly declined over the study period. Following reinfestation on day 28, neither sarolaner nor afoxolaner reached 90% efficacy by 48 h. By 72 h, sarolaner efficacy was 97.4% and afoxolaner efficacy was 86.3%. Only lotilaner achieved ≥ 90% efficacy by 24 h post-treatment and 24 h postinfestation on days 21 and 28. Time to ≥ 90% efficacy following new infestations consistently occurred 24–48 h earlier for lotilaner compared with sarolaner or afoxolaner. Conclusions Credelio (lotilaner) has a more rapid onset of acaricidal activity against A. americanum than Simparica Trio (sarolaner-moxidectin-pyrantel) and NexGard (afoxolaner). Only lotilaner’s speed of tick kill is sustained throughout the dosing period. Graphical Abstract

Background To manage tick infestations and reduce tick-borne pathogen transmission risk to dogs, compliant administration of a fast-acting ectoparasiticide is necessary. Isoxazoline-containing ectoparasiticide products provide systemic whole-body coverage; however, differences in tick kill have been observed between products and these differences may be more pronounced when controlling common dose-limiting tick species such as Amblyomma americanum . Methods Dogs were ranked by tick carrying capacity, randomly allocated to one of three treatment groups, and administered Bravecto® Chews (minimum 25 mg/kg fluralaner), Simparica TRIO® (minimum 1.2 mg/kg sarolaner, 24 µg/kg moxidectin, 5 mg/kg pyrantel), or no treatment. Dogs were infested with approximately 50 unfed adult (25 female, 25 male) A. americanum on days −2, 21, 28, and 35. Live tick counts were performed at 8, 12, 24, 48, and 72 h post-treatment (day 0) and post-infestation on days 21, 28, and 35. At each tick count timepoint, product efficacy was determined by comparing geometric mean live tick counts for each product-treated group to the untreated group and a linear mixed model was used for between-group comparisons. Results Compared with untreated dogs, significant control of existing A. americanum infestations began by 8 h post-treatment (81.6%) and reached 98.0% control by 12-h for Bravecto®-treated dogs. In comparison, significant control for Simparica TRIO®-treated dogs began by 24 h post-treatment (97.7%). When reinfested on day 21, A. americanum infestations were controlled more quickly for Bravecto® compared with Simparica TRIO®-treated dogs at 12 h (efficacy 95.3% versus 25.5%, P <  0.001) and 24 h (efficacy 99.7% versus 70.9%, P  < 0.001) post-infestation. Similarly, when reinfested on day 28, faster A. americanum control occurred for Bravecto® compared with Simparica TRIO®-treated dogs at 12 h (efficacy 87.9% versus 18.3%, P  < 0.001) and at 24 h (99.2% versus 59.3%, P  < 0.001) post-infestation. Finally, when reinfested on day 35, time to ≥ 90% efficacy was achieved by 48 h for Bravecto®-treated dogs compared with 72 h post-infestation for Simparica TRIO®-treated dogs. Both products performed within label indications and no treatment-related adverse reactions occurred during the study. Conclusions Amblyomma americanum infestations are controlled more quickly immediately upon treatment and at 21, 28, and 35 days post-treatment for Bravecto® compared with Simparica TRIO®-treated dogs. Graphical Abstract

Background Compliant ectoparasiticide product use is a comprehensive way to control ticks and reduce the risk of tick-borne pathogen transmission to dogs. Because the systemically acting isoxazoline ectoparasiticides require tick attachment for drug delivery, fast speed of kill is essential to minimize tick-borne pathogen transmission risk. Methods Dogs of satisfactory tick-carrying capacity were randomly allocated to treatment groups and administered, per label instructions, Bravecto ® Chews (minimum 25 mg/kg fluralaner), Simparica TRIO ® (minimum 1.2 mg/kg sarolaner, 24 µg/kg moxidectin, 5 mg/kg pyrantel), or no treatment. Dogs were infested with approximately 50 unfed adult (35 female, 15 male) Ixodes scapularis on Day -2, 21 and 28. Live tick counts were performed at 4, 8, 12 and 24 h post-treatment (Day 0) and post-infestation on Day 21 and 28. Tick control efficacy was determined by comparing live tick means for each product-treated group to the untreated control group and each other at all time points using a linear mixed model. The percent of dogs free of live ticks was analyzed using the Fisher’s exact test for treatment group comparison. Results The untreated control group maintained adequate tick infestations throughout the study. Using geometric means, an existing I. scapularis infestation was controlled by 99.7% and 93.0% 12 h post-treatment and by 100% and 99.5% 24 h post-treatment, for Bravecto ® and Simparica TRIO ® -treated dogs, respectively. Ixodes scapularis infestations were controlled more quickly for Bravecto ® - compared to Simparica TRIO ® -treated dogs on Day 21 at 8 h (efficacy 74.0% vs. 0.0%, p  = 0.003) and 12 h (efficacy 99.2% vs. 39.4%, p  < 0.001) post-infestation and Day 28 at 8 h (efficacy 92.2% vs. 0.0%, p  < 0.001) and 12 h (efficacy 99.6% vs. 27.7%, p  < 0.001) post-infestation. On Day 28 post-treatment, the efficacy of Bravecto ® and Simparica TRIO ® to control a new I. scapularis infestation was 100% and 96.6%, respectively, by 24 h post-infestation. Of product-treated dogs, 100% of Bravecto ® -treated dogs were free of live ticks by 24 h post-treatment or post-infestation. No treatment-related adverse reactions occurred during the study. Conclusions Ixodes scapularis infestations are controlled more quickly 21 and 28 days post-treatment for dogs administered a single dose of Bravecto ® compared to dogs administered a single dose of Simparica TRIO ® . Graphical Abstract