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BackgroundThe 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.MethodsOur 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.ResultsMetabolism 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.ConclusionsThese 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.

Abstract Background The speed with which acaricides paralyze and kill ticks is relevant to impeding pathogen transmission. The objective of this study was to assess early-onset lotilaner effects on the motility and weights of Amblyomma americanum ticks collected from treated dogs. Methods Twelve healthy dogs were randomized between two groups to receive either lotilaner (Credelio®) on Day 0 or to be sham treated. On Day 7, 25 male and 25 female A. americanum were placed under bandages, two on each flank of each dog. After 30 or 45 min, all unattached ticks were removed and T = 0 was set. At T = 2, 4, 8 and 24 h post attachment, 5 attached ticks removed from each bandage on each dog were weighed, assessed by blinded observers for righting ability and movement recorded. Results After the infestation period significantly fewer treated than control dogs had 20 ticks attached (50.0% versus 91.7%, P = 0.0015). At 24 h post attachment, mean weights of ticks from treated dogs (males 1.69 mg; females 2.72) were significantly less than ticks from controls (males 2.66 mg; females 4.67) (P male = 0.0002; P female

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.

Among the Ctenocephalides felis felis-borne pathogens, Bartonella henselae, the main aetiological agent of cat scratch disease (CSD), is of increasing comparative biomedical importance. Despite the importance of B. henselae as an emergent pathogen, prevention of the diseases caused by this agent in cats, dogs and humans mostly relies on the use of ectoparasiticides. A vaccine targeting both flea fitness and pathogen competence is an attractive choice requiring the identification of flea proteins/metabolites with a dual effect. Even though recent developments in vector and pathogen -omics have advanced the understanding of the genetic factors and molecular pathways involved at the tick-pathogen interface, leading to discovery of candidate protective antigens, only a few studies have focused on the interaction between fleas and flea-borne pathogens. Taking into account the period of time needed for B. henselae replication in flea digestive tract, the present study investigated flea-differentially abundant proteins (FDAP) in unfed fleas, fleas fed on uninfected cats, and fleas fed on B. henselae-infected cats at 24 hours and 9 days after the beginning of blood feeding. Proteomics approaches were designed and implemented to interrogate differentially expressed proteins, so as to gain a better understanding of proteomic changes associated with the initial B. henselae transmission period (24 hour timepoint) and a subsequent time point 9 days after blood ingestion and flea infection. As a result, serine proteases, ribosomal proteins, proteasome subunit α-type, juvenile hormone epoxide hydrolase 1, vitellogenin C, allantoinase, phosphoenolpyruvate carboxykinase, succinic semialdehyde dehydrogenase, glycinamide ribotide transformylase, secreted salivary acid phosphatase had high abundance in response of C. felis blood feeding and/or infection by B. henselae. In contrast, high abundance of serpin-1, arginine kinase, ribosomal proteins, peritrophin-like protein, and FS-H/FSI antigen family member 3 was strongly associated with unfed cat fleas. Findings from this study provide insights into proteomic response of cat fleas to B. henselae infected and uninfected blood meal, as well as C. felis response to invading B. henselae over an infection time course, thus helping understand the complex interactions between cat fleas and B. henselae at protein levels.

Abstract Background Point-of-care ultrasound (POCUS) has been used in various vascular access contexts; however, to date, it has not been widely adopted in haemodialysis clinics. People with end-stage kidney disease receiving haemodialysis require an arteriovenous fistula (AVF), arteriovenous graft (AVG), or central venous access device (CVAD) in order to access their blood for therapy/treatment. Cannulation issues, such as haematoma and extravasation, related to AVFs and AVGs are common. This pilot and feasibility study will assess the feasibility of a randomised controlled trial aimed at evaluating whether POCUS-guided cannulation results in more successful and accurate AVF needle placement than the standard practice of blind cannulation. Methods A controlled, random order crossover design will be used to evaluate two clinical conditions: (1) POCUS-guided cannulation and (2) standard practice of blind cannulation, when used by haemodialysis nurses. The feasibility of conducting this type of trial for these two clinical conditions will be assessed through recruitment, retention, and attrition rates; perceptions of acceptability; implementation measures; and assessment of methods of data collection. Clinical outcomes to be assessed are overall cannulation success on first attempt, accuracy of needle tip placement, number of extravasations, procedural time, and patient and nurse perceptions. The setting is a 16-chair dialysis unit in regional Australia. Participants will include adult haemodialysis patients with an AVF in situ for greater than 2 months and haemodialysis-trained registered nurses working full- or part-time. Clinical outcomes will be analysed using generalised linear mixed models. Feasibility data will be reported using descriptive statistics. Qualitative audio data will be digitally recorded, transcribed verbatim, and analysed using thematic analysis. Discussion Point-of-care ultrasound for cannulation has the potential to promote high-quality, safe nursing care and decrease cannulation damage for patients on haemodialysis. Due to the lack of evidence for patient benefit and its innovative and niche use in haemodialysis centres, POCUS is currently only specified in one international guideline. This study will inform sample size calculations for a future multi-site trial. Trial registration Australian New Zealand Clinical Trials Registry, (21/11/2017) ACTRN12617001569392

Abstract Background An investigation was conducted in West Central Florida, USA to evaluate the efficacy of either topically applied fluralaner or topically applied selamectin to control flea infestations, minimize dermatologic lesions and reduce pruritus in naturally flea infested cats over a 12-week period. When dogs were present in the households, they were treated with either oral fluralaner (if household cats were treated with topical fluralaner) or oral sarolaner (if household cats were treated with topical selamectin). Methods Thirty-one cats in 20 homes were treated once with fluralaner topical solution on day 0 and 18 dogs in these homes were administered a single fluralaner chewable. Twenty-nine cats in 18 homes were treated once monthly with a selamectin topical solution for 3 treatments and 13 dogs in these same homes were treated once monthly for 3 treatments with a sarolaner chewable. Fleas on cats were counted by flea combing, fleas on dogs were estimated using visual area counts and fleas in the indoor premises were assessed using intermittent-light flea traps. Blinded-assessments of feline dermatologic lesions were conducted monthly and pruritus severity was evaluated by pet owners. Results A single topical application of fluralaner reduced flea populations on cats by 96.6% within 7 days and by 100% at 12 weeks post-treatment. This efficacy was significantly greater than selamectin treatment where single topical application reduced flea populations on cats by 79.4% within 7 days of initial treatment and 3 consecutive monthly treatments reduced flea populations by 91.3% at the end of 12 weeks. At the end of the 12-week study, all fluralaner-treated cats were flea-free and this was significantly greater than the 38.5% of selamectin treated cats that were flea-free. At the end of the study, fleas were completely eradicated (from cats, dogs and homes) in 95.0% of fluralaner treatment group homes, significantly greater than the 31.3% of selamectin/sarolaner treatment group homes with complete flea eradication. Owner reported cat pruritus was reduced similarly in both treatment groups. Significant improvements in dermatologic lesion scores were achieved by day 30 in fluralaner treated cats and by day 60 in selamectin treated cats. Conclusions An in-home investigation in subtropical Florida found that 1 application of topical fluralaner eliminated flea infestations on cats and in homes significantly more effectively than 3 consecutive monthly doses of selamectin.

Abstract Background An in-home investigation of naturally flea infested dogs was conducted in West Central Florida, USA to evaluate and compare the effectiveness of two different oral flea adulticides to control flea infestations, minimize dermatologic lesions and reduce pruritus over an 8-week period. Methods Twenty-nine dogs living in 19 homes and another 26 dogs residing in 16 different homes were orally administered either a sarolaner or spinosad chewable, respectively on day 0 and once between days 28–30. Products were administered by study personnel according to label directions. Flea populations on dogs were estimated using visual area counts and flea infestations in the indoor premises were assessed using intermittent-light flea traps on days 0, 7, 14, 21 and once between days 28–30, 40–45, and 56–60. Assessments of dermatologic lesions were conducted monthly during the study and severity of pruritus was evaluated throughout the study on the same schedule as flea counts were conducted. Concurrent treatments for existing skin disease were not allowed. Results The administration of sarolaner or spinosad reduced flea populations on dogs by 99.0% and 97.3%, respectively within 7 days. Flea infestations on the sarolaner- and spinosad-treated dogs were reduced by > 99% at every counting period from day 14 post-treatment through the end of the 8-week study. At the end of the study 96.4 and 92.0% of the dogs treated with sarolaner and spinosad, respectively were flea-free. Flea populations in the indoor premises were also markedly reduced the end of the study, with 100 and 99.8% reductions in flea trap counts in the sarolaner and spinosad treatment groups, respectively. FAD lesion scores, atopic dermatitis lesions scores (CADESI-4) and pruritus severity scores were also markedly improved with both formulations. Conclusions An in-home clinical field study conducted during the summer of 2016 in subtropical Florida demonstrated that two-monthly administrations of either sarolaner or spinosad chewables almost completely eliminated flea infestations on dogs and in private residences, while markedly reducing dermatology lesions and pruritus.

Abstract Background Fluralaner is a potent insecticide and acaricide with rapid and persistent efficacy. This study measured the efficacy of fluralaner flavored chews (Bravecto®, Merck Animal Health) administered to dogs against adult Ctenocephalides felis felis and egg production. Methods Twelve purpose-bred dogs were randomly allocated to two groups of six dogs each. Dogs in treatment group 1 were administered a single fluralaner flavored chew to achieve a minimum dose of at least 25 mg/kg while treatment group 2 served as untreated controls. On Days −2, 28, 56, 84, 91, 98, 105, 112, and 120 post-treatment, each dog was infested with approximately 200 unfed cat fleas, C. felis felis (KS1 strain). Forty-eight hours after treatment and 48 h after each infestation, eggs were collected over a 3-h period, counted and viability determined. Dogs were combed to remove any remaining fleas. Results Treatment of dogs with oral fluralaner provided a 100 % reduction in flea counts 48 h after treatment and within 48 h of every post-treatment infestation through Day122. Egg production from fluralaner treated dogs was reduced by 99.9 % (two eggs from one dog) within 48 h after treatment and not a single egg (100 % efficacy) was thereafter collected from treated dogs. Adult flea counts and egg production from the fluralaner-treated dogs were significantly lower than for non-treated controls at all post-treatment evaluations (P

Abstract Background A study was conducted to evaluate the effectiveness of afoxolaner chewables to control flea populations in naturally infested dogs in private residences in Tampa FL, USA. Evaluations of on-animal and premises flea burdens, flea sex structure and fed-unfed premises flea populations were conducted to more accurately assess flea population dynamics in households. Methods Thirty seven naturally flea infested dogs in 23 homes in Tampa, FL were enrolled in the study and treated with afoxolaner chewables. Chewables (NexGard® Chewables; Merial) were administered according to label directions by study investigators on study day 0 and once again between study days 28 and 30. Flea infestations on pets were assessed using visual area thumb counts and premises flea infestations were assessed using intermittent-light flea traps on days 0, 7, 14, 21, and once between study days 28–30, 40–45, and 54–60. Results Within 7 days of administration of afoxolaner chewable tablets, flea counts on dogs were reduced by 99.3 %. By one month post-treatment, total flea counts on dogs were reduced by 99.9 %, with 97.3 % (36/37) of the dogs being flea free. Following the second dosing on study day 28–30, total on-dog flea burden was reduced by 100 % on days 40–45 and 54–60. On day 0, the traps collected a geometric mean of 18.2 fleas. Subsequent reductions in emerging flea populations were 97.7 and 100 % by days 28–30 and 54–60, respectively. There were 515 total fleas (Ctenocephalides felis felis) collected in the intermittent light flea traps on day 0, and 40.4 % of those fleas displayed visual evidence of having fed. Seven days after initial treatment, only 13.1 % of the fleas contained blood and by day 14 only 4.9 % of the fleas collected in traps displayed evidence of having fed. On day 0, prior to treatment, 60 % of the unfed fleas collected in intermittent-light flea traps were females, but by days 28–30, unfed males accounted for 78 % of the population. Conclusions This in-home investigation conducted during the summer of 2014 in subtropical Tampa, FL demonstrated that afoxolaner chewables rapidly and effectively eliminated flea populations in infested dogs and homes.

Presents an industry insider's viewpoint on what effect the downfall of the "dot-coms" may have for educational technology and for schools using Internet services. Includes some key sign posts that point toward long-term success for these entrepreneurial education firms and for schools. Offers a checklist of what educators say they want from Internet companies. (GR)