Retrospective analysis of canine fecal flotation and coproantigen immunoassay hookworm positive results in Greyhounds and other dog breeds.

Recent studies demonstrated that Greyhounds are commonly infected with Ancylostoma caninum and these infections have been shown to be resistant to anthelmintics. This study evaluated samples submitted to a commercial reference laboratory (IDEXX Laboratories) for canine fecal flotation zinc sulfate centrifugation and coproantigen immunoassay between January 1, 2019, and July 30, 2023 for evidence that Greyhounds were more often positive for Ancylostoma spp. (hookworms) compared to other breeds. The purpose of the study was to determine if Greyhounds were more likely to be hookworm-positive compared to other breeds, if Greyhounds on preventives with efficacy against hookworm infections are more likely to test positive than other breeds, if their infections take longer to resolve, to estimate how long this takes and to assess whether the proportion of hookworm positive tests for all breeds is increasing over time. Records of 25,440,055 fecal results were obtained representing 17,671,724 unique dogs. Of these, 49,795 (∼0.3%) were Greyhounds. The overall odds ratio (OR) of 15.3 (p < 0.001) suggests that Greyhounds are at significantly higher risk than other breeds for hookworm positive float findings, and the OR of 14.3 (p < 0.001) suggests significantly higher risk for hookworm antigen positive results. The median time to negative testing event from the Turnbull distribution estimate was in the interval of 1-2 days for other breeds and 71-72 days for Greyhounds. These results provide evidence that anthelmintic resistant A. caninum strains may be having population-level impacts on the frequency and duration of infections in Greyhounds. The findings have broader health implications beyond Greyhounds as MADR A. caninum strains could spread to other breeds and even pet owners.

vivoBodySeg: Machine learning-based analysis of C. elegans immobilized in vivoChip for automated developmental toxicity testing.

Developmental toxicity (DevTox) tests evaluate the adverse effects of chemical exposures on an organism's development. While large animal tests are currently heavily relied on, the development of new approach methodologies (NAMs) is encouraging industries and regulatory agencies to evaluate these novel assays. Several practical advantages have made C. elegansa useful model for rapid toxicity testing and studying developmental biology. Although the potential to study DevTox is promising, current low-resolution and labor-intensive methodologies prohibit the use of C. elegans for sub-lethal DevTox studies at high throughputs. With the recent availability of a large-scale microfluidic device, vivoChip, we can now rapidly collect 3D high-resolution images of ~ 1,000 C. elegans from 24 different populations. In this paper, we demonstrate DevTox studies using a 2.5D U-Net architecture (vivoBodySeg) that can precisely segment C. elegans in images obtained from vivoChip devices, achieving an average Dice score of 97.80. The fully automated platform can analyze 36 GB data from each device to phenotype multiple body parameters within 35 min on a desktop PC at speeds ~ 140x faster than the manual analysis. Highly reproducible DevTox parameters (4-8% CV) and additional autofluorescence-based phenotypes allow us to assess the toxicity of chemicals with high statistical power.

OrganoidChip facilitates hydrogel-free immobilization for fast and blur-free imaging of organoids.

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids' viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.

Use of a Point-of-Care Progesterone Assay to Predict Onset of Parturition in the Bitch.

An assay of circulating progesterone (P4) is commonly used to estimate progress through late gestation in the bitch. Point-of-care assays provide rapid results, a major advantage over laboratory-based assays. This study aims to compare P4 levels determined by the Catalyst® Progesterone point-of-care assay with those determined by chemiluminescent immunoassay (CLIA) and to identify the expected distribution of Catalyst P4 levels at time intervals 3 days prior to the onset of parturition in pregnant bitches. Twenty-eight pregnant bitches carrying two or more fetuses were admitted to a specialist veterinary reproduction hospital 53 days after the onset of cytological diestrus or, when that date was not known, 57 days after the last mating. Vaginal speculum examinations were performed every 6 h until the onset of cervical dilatation (TCD). Serum samples were collected twice daily (08h00 and 18h00) until TCD. For most samples, fresh serum was assayed for P4 immediately using the Catalyst assay (CatP4), then frozen until assayed by CLIA (IMMULITE 2000; ImmP4). However, for some samples, CatP4 was not analyzed prior to freezing. For these data points (n = 33), CatP4 for fresh serum was estimated from CatP4 assayed on frozen-thawed serum, based on a comparison between CatP4 on fresh vs. frozen-thawed sera. In comparison to ImmP4, CatP4 levels up to and including 7 nmol/L appear to have a constant bias of -1.69 nmol/L (limits of agreement -4.91 to 1.52), while levels >7 nmol/L appear to have a proportional bias of -17.9% (limits of agreement -68.6% to 32.7%). Bootstrapped percentiles of CatP4 results spanned 0.4-9 nmol/L within 12 h of TCD, 0.9-11 nmol/L 12-24 h from TCD, and 2.2-13.5 nmol/L 24-36 h from TCD. A CatP4 >9 nmol/L indicates a bitch that is unlikely to reach TCD within 12 h. Bitches with CatP4s below 3.5 nmol/L are likely to reach TCD within 36 h and bitches with a CatP4 below 2.2 nmol/L are likely to reach TCD within 24 h. In conclusion, the Catalyst Progesterone assay provides rapid assessment of circulating P4 in the bitch, with clinical application in the monitoring of late term pregnant bitches.

Detection of Giardia and helminths in Western Europe at local K9 (canine) sites (DOGWALKS Study).

BACKGROUND: Intestinal parasite contamination from infected dogs can place other dogs and humans at risk. A study was initiated to estimate the prevalence of canine intestinal parasitism by collecting fecal samples in cities across Western Europe. METHODS: Fresh fecal samples were collected from 2469 dogs visiting 164 parks in 33 cities across 12 countries. Each owner responded to a questionnaire focusing on their dog's signalment and recent anthelmintic treatment history. The collected samples were examined for hookworms, whipworms, ascarids and Giardia using a coproantigen diagnostic immunoassay and microscopy following centrifugal flotation. RESULTS: Nematodes or Giardia were detected in at least one sample from 100% of cities and in 93.3% of parks. Nematodes were detected in 57% of parks. Overall, 22.8% of dogs tested positive for an intestinal parasite, with Giardia being the most commonly identified parasites (17.3% of dogs, 83.5% of parks). For nematode infection, 7.6% of all dogs tested positive, with 9.9% of dogs aged < 1 year infected, 7.7% of those aged 1-3 years, 7.3% of those aged 4-6 years and 6.6% of those aged ≥ 7 years. Among the nematodes detected, ascarids were the most prevalent (3.6% of dogs, parks, 28.7% of parks), being most common in dogs aged < 1 year but also present in older dogs, including those aged ≥ 7 years. Hookworms and whipworms were detected in 3.2% and 2.3% of dogs of all ages, respectively, and in 37.2% and 17.7% of parks, respectively. A larger proportion of fecal samples tested positive with the coproantigen immunoassay than with centrifugal flotation. Positive test results for Giardia were sevenfold higher when both diagnostic tests were used than when centrifugal flotation alone was used, and there were 60% more positive test results for nematodes when both tests were used than when flotation alone was used. Overall, 77.2% of owners reported previous anthelmintic treatment, among whom at least 62.7% failed to follow recommended treatment frequency. Dogs receiving anthelmintic within the previous month had a lower percentage of nematode infection than those in which > 1 month had passed since the previous dose. CONCLUSIONS: The prevalence estimates of intestinal parasite infections in dogs reported here highlight the need for owner education concerning guidelines for regular testing and treatment, even in older dogs. Failure to adhere to guidelines can result in ongoing transmission of these infections, including those with zoonotic potential. Combining coproantigen immunoassay with centrifugal flotation for diagnostic testing and regular anthelmintic treatment are important measures for ensuring optimal intestinal parasite control.

Seropositivity of main vector-borne pathogens in dogs across Europe.

BACKGROUND: Canine vector-borne disease (CVBD) has been an area of increasing interest in Europe over the last few decades, and there have been changes in the prevalence and distribution of many of these diseases. Monitoring CVBD infections in Europe is often done by individual countries, but aggregated data for the European countries are helpful to understand the distribution of CVBDs. METHODS: We used an extensive retrospective database of results from point-of-care rapid enzyme-linked immunosorbent assay (ELISA) tests on dogs across Europe to identify distribution and seropositivity in animals tested for selected CVBDs (Anaplasma spp., Ehrlichia spp., Borrelia burgdorferi, Leishmania spp., and Dirofilaria immitis) from 2016 through 2020. Geographic distribution of positive tests and relative percent positive values were mapped by the Nomenclature of Territorial Units for Statistics classification for regions with sufficient test results for reporting. RESULTS: A total of 404,617 samples corresponding to 1,134,648 canine results were available from dogs tested in 35 countries over the 5-year study period. Over this period the number of test results per year increased whereas test positivity decreased. Leishmania spp. had the largest increase in total test results from 25,000 results in 2016 to over 60,000 results in 2020. Test positivity for Leishmania spp. fell from 13.9% in 2016 to 9.4% in 2020. Test positivity fell for Anaplasma spp. (7.3 to 5.3%), Ehrlichia spp. (4.3 to 3.4%), and Borrelia burgdorferi (3.3 to 2.4%). Dirofilaria immitis test positivity trended down with a high of 2.7% in 2016 and low of 1.8% in 2018. Leishmania spp. test positivity was highest in endemic areas and in several non-endemic countries with low numbers of test results. Co-positivity rates were significantly higher than expected for all pathogen test positive pairs except for Ehrlichia spp. with Borrelia burgdorferi and D. immitis with Borrelia burgdorferi. CONCLUSIONS: This study represents the largest data set on CVBD seropositivity in Europe to date. The increase in the number of test results and decreasing test positivity over the study period may reflect changes in testing behavior and increased screening of healthy animals. The Europe-wide mapping of CVBD provides expected test positivity that can help inform veterinarians' decisions on screening and improve prevention and identification of these important, sometimes zoonotic, diseases.

A 3-year retrospective analysis of canine intestinal parasites: fecal testing positivity by age, U.S. geographical region and reason for veterinary visit.

BACKGROUND: Canine life stage is a key factor in parasite prevalence as clinical signs associated with parasitism are more common in pups. In adult dogs, health status and geographical region may also play a role in parasite prevalence. The purpose of this study was to evaluate fecal test results using zinc sulfate flotation by centrifugation combined with fecal antigen testing for hookworms (Ancylostoma spp. Uncinaria stenocephala), ascarids (Toxocara canis, Toxascaris spp., Baylisascaris spp.) and whipworms (Trichuris vulpis) sorted by age, geographical region and veterinary visit type. METHODS: A retrospective sample of intestinal parasite panels submitted to IDEXX Laboratories from 1,626,104 individual dogs were selected from the continental USA from 1 January 2017 to 31 December 2019. These data contain results from fecal exams performed using zinc sulfate flotation by centrifugation paired with coproantigen immunoassay results for hookworms, ascarids, whipworms and Giardia (Fecal Dx® with Giardia coproantigen immunoassay plate). For paired testing, if either the coproantigen assay or flotation test was positive, the sample was considered to be positive. Data were summarized by age category, U.S. Census Bureau geographical region (Northeast, South, Midwest, West) and veterinary visit type. Visit types were subdivided into Wellness Visits and Other Clinical Visits in which a fecal sample was submitted. RESULTS: In dogs presenting for either Wellness Visits or Other Clinical Visits in which Giardia testing was included, Giardia had the highest positivity (combined results for microscopy and coproantigen: 12.2 and 10.8%, respectively), followed by hookworms (combined microscopy and coproantigen: 4.1 and 4.2%, respectively), ascarids (combined microscopy and coproantigen: 2.5 and 1.7%, respectively) and whipworms (combined microscopy and coproantigen: 1.1 and 1.4%, respectively). When all test results were pooled together, pups aged 2-6 months were observed to have the highest proportion of positive results by either microscopy or coproantigen immunoassay regardless of clinical visit type. Parasite positivity varied by geographical region. Regardless of visit type, age or geographical region, the coproantigen method was observed to find a higher proportion of positive test results than microscopy in Giardia, ascarids, hookworms and whipworms. CONCLUSIONS: The Fecal Dx® coproantigen immunoassay combined with the zinc sulfate flotation by centrifugation method uncovers a higher number of positive hookworm, ascarid and whipworm infections than zinc sulfate flotation alone in both pups and adult dogs across all geographical regions of the USA regardless of visit type.

A Meta-Analysis of Blood Pressure Disparities Among Sexual Minority Men.

Purpose: The aim was to review and summarize reports on three measures of elevated blood pressure (BP) among sexual minority men (encompassing men who have sex with men [MSM] and both men and women [MSMW]), using men who have sex with women (MSW) as the reference population. Methods: Crude prevalence rates were calculated, and a meta-analysis was conducted to summarize the likelihood of elevated BP history, antihypertensive medication use, and elevated BP above a cutoff value, as well as the mean differences (MDs) in systolic BP (SBP) and diastolic BP (DBP) measurements. We used random effects to generate estimates with their respective 95% confidence intervals (CIs); alpha was set at 0.05. Results: Studies (n = 20) were published between 2007 and 2018, mostly in the United States. The likelihood of elevated BP history was not statistically significantly higher among sexual minority men, except when the measurement of sexual orientation was multidimensional (odds ratio [OR] 1.41, 95% CI 1.12-1.78). The likelihood of antihypertensive medication use was only statistically significantly higher for men who self-identified as MSMW (OR 1.44, 95% CI 1.11-1.85). When elevated BP was determined through a set cutoff, MSM were less likely (OR 0.34, 95% CI 0.16-0.70), whereas MSMW were more likely (OR 2.25, 95% CI 1.54-3.28) to have elevated BP. Although there were no statistically significant findings in the MD for SBP, the MD for DBP among sexual minority men was significantly higher (MD 1.46, 95% CI 1.38-1.55 mmHg) than among the MSW comparison group. Conclusions: Sexual minority men classified using a multidimensional approach to sexual orientation had a significantly higher likelihood of elevated BP history. Using BP cutoffs yielded opposite effects in MSM and MSMW. Although SBP was not different compared to MSW, DBP-a marker of hypertension at earlier ages-was elevated among sexual minority men.

Symmetric dimethylarginine concentrations in healthy neonatal foals and mares.

BACKGROUND: Symmetric dimethylarginine (SDMA) is a renal biomarker correlated with glomerular filtration rate (GFR). OBJECTIVES: Describe changes in SDMA in clinically healthy foals and their mares during the first month postfoaling. ANIMALS: Convenience sampling of healthy periparturient Thoroughbred mares and their full-term foals from a population of client-owned horses. METHODS: Serum and EDTA whole blood samples were collected from mares in their last month of pregnancy and then from mares and foals at approximately <12 hours, 48 hours, 7 days, and 30 days postbirth. Samples were processed at a commercial reference laboratory for CBC and serum biochemistry, including SDMA concentrations. RESULTS: A total of 125 foals and 104 mares were included. Upper limits for SDMA concentrations in foals were above the adult horse reference interval for the first 20 or more days of life. Median SDMA concentrations decreased from 70 µg/dL (range, 7-100 µg/dL) to 18 µg/dL (range, 6-27 µg/dL) during the first 3 to 4 weeks of life. At birth, the SDMA concentration reference range was established as 0 to 100 µg/dL (upper limit of the assay); 0 to 85 µg/dL for 1 to 4 days old, 0 to 36 µg/dL for 5 to 10 days old, and 0 to 24 µg/dL for 20 to 30 days old. The upper reference limits for SDMA concentrations in mares did not differ from the general reference interval for adult horses. No correlation was identified between mare and foal SDMA concentrations (ρ = .06, P = .58). CONCLUSIONS AND CLINICAL IMPORTANCE: Foal SDMA concentrations remained higher than the upper limit of the adult reference range and foals require a different reference range dependent on age.

Retrospective analysis of feline intestinal parasites: trends in testing positivity by age, USA geographical region and reason for veterinary visit.

BACKGROUND: The goals of this retrospective study were to estimate parasite positivity in samples from cats using zinc sulfate fecal flotation by centrifugation ("centrifugation") and coproantigen and examine trends with age, geographical region and reason for visit to veterinarian. Common methods of parasite detection, such as centrifugal flotation, passive flotation, or direct smear, may underrepresent the true prevalence of intestinal parasites in cats. Coproantigen testing detects more positive samples than traditional methods alone. METHODS: Feline fecal test results from the continental USA containing results for fecal exams performed using centrifugation paired with coproantigen results for ascarid, hookworm, whipworm and Giardia were obtained from the database of a national commercial reference laboratory comprised of multiple regional sites. RESULTS: Parasite positivity was highest in samples from young cats and decreased with cat age. The western region of the USA had lower total parasite positivity than other regions for all parasites except Giardia. Cats receiving fecal tests during veterinary wellness visits had only slightly lower parasite positivity than samples from cats during sick clinical visits. CONCLUSIONS: This study showed a larger population of cats are at increased risk of parasitism than commonly believed and coproantigen testing produces more positive test results for the four parasites that antigen can detect than centrifugation of feline fecal samples.

An automated microfluidic device for time-lapse imaging of mouse embryonic stem cells.

Long-term, time-lapse imaging studies of embryonic stem cells (ESCs) require a controlled and stable culturing environment for high-resolution imaging. Microfluidics is well-suited for such studies, especially when the media composition needs to be rapidly and accurately altered without disrupting the imaging. Current studies in plates, which can only add molecules at the start of an experiment without any information on the levels of endogenous signaling before the exposure, are incompatible with continuous high-resolution imaging and cell-tracking. Here, we present a custom designed, fully automated microfluidic chip to overcome these challenges. A unique feature of our chip includes three-dimensional ports that can connect completely sealed on-chip valves for fluid control to individually addressable cell culture chambers with thin glass bottoms for high-resolution imaging. We developed a robust protocol for on-chip culturing of mouse ESCs for minimum of 3 days, to carry out experiments reliably and repeatedly. The on-chip ESC growth rate was similar to that on standard culture plates with same initial cell density. We tested the chips for high-resolution, time-lapse imaging of a sensitive reporter of ESC lineage priming, Nanog-GFP, and HHex-Venus with an H2B-mCherry nuclear marker for cell-tracking. Two color imaging of cells was possible over a 24-hr period while maintaining cell viability. Importantly, changing the media did not affect our ability to track individual cells. This system now enables long-term fluorescence imaging studies in a reliable and automated manner in a fully controlled microenvironment.

Line excitation array detection fluorescence microscopy at 0.8 million frames per second.

Three-dimensional, fluorescence imaging methods with ~1 MHz frame rates are needed for high-speed, blur-free flow cytometry and capturing volumetric neuronal activity. The frame rates of current imaging methods are limited to kHz by the photon budget, slow camera readout, and/or slow laser beam scanners. Here, we present line excitation array detection (LEAD) fluorescence microscopy, a high-speed imaging method capable of providing 0.8 million frames per second. The method performs 0.8 MHz line-scanning of an excitation laser beam using a chirped signal-driven longitudinal acousto-optic deflector to create a virtual light-sheet, and images the field-of-view with a linear photomultiplier tube array to generate a 66 × 14 pixel frame each scan cycle. We implement LEAD microscopy as a blur-free flow cytometer for Caenorhabditis elegans moving at 1 m s-1 with 3.5-µm resolution and signal-to-background ratios >200. Signal-to-noise measurements indicate future LEAD fluorescence microscopes can reach higher resolutions and pixels per frame without compromising frame rates.

High-Content Microfluidic Screening Platform Used To Identify σ2R/Tmem97 Binding Ligands that Reduce Age-Dependent Neurodegeneration in C. elegans SC_APP Model.

The nematode Caenorhabditis elegans, with tractable genetics and a well-defined nervous system, provides a unique whole-animal model system to identify novel drug targets and therapies for neurodegenerative diseases. Large-scale drug or target screens in models that recapitulate the subtle age- and cell-specific aspects of neurodegenerative diseases are limited by a technological requirement for high-throughput analysis of neuronal morphology. Recently, we developed a single-copy model of amyloid precursor protein (SC_APP) induced neurodegeneration that exhibits progressive degeneration of select cholinergic neurons. Our previous work with this model suggests that small molecule ligands of the sigma 2 receptor (σ2R), which was recently cloned and identified as transmembrane protein 97 (TMEM97), are neuroprotective. To determine structure-activity relationships for unexplored chemical space in our σ2R/Tmem97 ligand collection, we developed an in vivo high-content screening (HCS) assay to identify potential drug leads. The HCS assay uses our recently developed large-scale microfluidic immobilization chip and automated imaging platform. We discovered norbenzomorphans that reduced neurodegeneration in our C. elegans model, including two compounds that demonstrated significant neuroprotective activity at multiple doses. These findings provide further evidence that σ2R/Tmem97-binding norbenzomorphans may represent a new drug class for treating neurodegenerative diseases.

A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans.

Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only.

Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model.

Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ∼4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ∼100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ∼1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.