Geographic Distribution of Human Infections with Zoonotic Ancylostoma ceylanicum and Anthropophilic Hookworms in Ecuador: A Retrospective Analysis of Archived Stool Samples.

Zoonotic human infections with Ancylostoma ceylanicum have recently been reported in the Americas. We used archived human stool samples to study the geographic distribution of human infections with A. ceylanicum and anthropophilic hookworms in different geoclimatic regions (coastal, Andean, and Amazon) of Ecuador. We analyzed retrospectively archived human stool samples from five studies previously screened for hookworm infection by microscopy, of which four included hookworm-positive samples only and one involved hookworm-negative samples to increase geographic distribution of sampling. Stools were analyzed using multi-parallel quantitative polymerase chain reaction (qPCR) assays to detect Necator americanus, Ancylostoma duodenale, A. ceylanicum, Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. Sequencing was done for the A. ceylanicum cox1 gene. A total of 132 samples were analyzed, of which 69 (52.3%) were from hookworm-positive and 63 (47.7%) from hookworm-negative individuals by microscopy. Overall, 82.6% of microscopy-positive samples and 33.3% of microscopy-negative samples were positive for hookworm by qPCR. Of microscopy-positive samples, 36.2% were A. ceylanicum, 37.7% A. duodenale, and 33.3% N. americanus, whereas equivalent proportions for microscopy-negative samples were 1.6%, 31.7%, and 1.6%, respectively. Ancylostoma duodenale was the most widely dispersed geographically, followed by N. americanus. Ancylostoma ceylanicum was least dispersed but was detected in coastal and Amazon regions. In conclusion, human infections with A. ceylanicum, A. duodenale, and N. americanus were detected in different geoclimatic regions of Ecuador. Additional studies are required to further define the epidemiology of human A. ceylanicum infections, but the potentially widespread presence of this helminth in human populations in Ecuador has implications for hookworm control strategies.

Quantification of circulating tumour cells over time in dogs with appendicular osteosarcoma.

Enumeration of circulating tumour cells (CTC) has shown promise for prognostication and guidance of therapeutic decisions in human cancers. The objective of this study was to enumerate CTC over time in dogs with naturally occurring osteosarcoma (OSA), and to determine correlation with patient outcome. Twenty-six dogs with OSA and no evidence of metastatic disease at the time of amputation were enrolled. Dogs were assessed for lung metastases and CTC prior to and following amputation, and at each chemotherapy visit. Twenty-one dogs completed the study. Nineteen dogs were euthanized and two were alive and free of metastases. Overall survival time ranged from 88 to 1058 days (median survival time (MST) 374 days). Increased serum alkaline phosphatase activity, advanced age, and higher body weight were significantly associated with lower MST. Dogs with OSA had a mean of 356 (0 to 4443) CTC/106 leukocytes. In 12 of 15 dogs that developed radiographic evidence of metastasis, a pre-metastatic CTC spike was retrospectively detectable on average 36.5 (1-100 days) days prior to metastasis and was associated with significantly shorter MST (301 ± 64 vs. 626 ± 55 days; p = .0107). In a multivariable analysis, dogs with a CTC spike were 10× more likely to die compared with those without. These results suggest that a spike in CTC frequency precedes detection of metastasis in dogs with OSA and is associated with shorter survival. More frequent enumeration of CTC in a larger cohort of dogs with OSA may be warranted.

Neuroangiostrongyliasis: Updated Provisional Guidelines for Diagnosis and Case Definitions.

Angiostrongylus cantonensis is the main causative agent for eosinophilic meningoencephalitis in humans. Larvae are rarely found in the cerebral spinal fluid (CSF). Consequently, serology and DNA detection represent important diagnostic tools. However, interpretation of the results obtained from these tools requires that more extensive accuracy studies be conducted. The aim of the present study is to update guidelines for diagnosis and case definitions of neuroangiostrongyliasis (NA) as provided by a working group of a recently established International Network on Angiostrongyliasis. A literature review, a discussion regarding criteria and diagnostic categories, recommendations issued by health authorities in China and an expert panel in Hawaii (USA), and the experience of Thailand were considered. Classification of NA cases and corresponding criteria are proposed as follows: minor (exposure history, positive serology, and blood eosinophilia); major (headache or other neurological signs or symptoms, CSF eosinophilia); and confirmatory (parasite detection in tissues, ocular chambers, or CSF, or DNA detection by PCR and sequencing). In addition, diagnostic categories or suspected, probable, and confirmatory are proposed. Updated guidelines should improve clinical study design, epidemiological surveillance, and the proper characterization of biological samples. Moreover, the latter will further facilitate accuracy studies of diagnostic tools for NA to provide better detection and treatment.

Teaching Accuracy through Repeated Gamified Echography Training (TARGET): Assessment of an Ultrasound Skill Simulator in Teaching Ultrasound-Guided Needle Placement, a Prospective Observational Study.

The increasing use of ultrasound in veterinary private practice and demand for skilled operators upon graduation has placed an increased burden on the ever-dwindling number of academic radiologists. Simulation-based medical education can help prepare for and consequently reduce this burden, allowing for the acquisition of clinical skills through deliberate practice in a safe, controlled, and low-stakes environment. Ultrasound-guided fine needle placement is the foundation for more advanced interventions such as ultrasound-guided fine needle aspirates and centeses. A reusable novel ultrasound skill simulator consisting of metal targets wired to a circuit and suspended in ballistics gel was created to teach ultrasound-guided fine needle placement. Forty-seven second-year veterinary students watched an instructional video and performed two ultrasound-guided fine needle placement skill tests on the simulator with a period of practice between. Significant improvement in time to task completion (p = .0021) was noted after the period of practice. The majority of student feedback was positive with 89% (42/47) indicating they would use the simulator again to practice and that it should be incorporated into the curriculum, 74% (35/47) indicating their basic skills, knowledge, and confidence using ultrasound improved using the simulator, and 55% (26/47) indicating they could now teach this skill to a peer. The authors suggest further development of this model for ease of manufacture and increased variation in difficulty, and veterinary curriculum incorporation for basic ultrasound-guided fine needle placement training.

Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus.

Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunophenotypes and cytokine responses to FLS stimulation is unknown. To address these questions, alveolar macrophages/monocytes (AMMs) were isolated from bronchoalveolar lavage fluid and MDMs derived from blood of six healthy horses. Separately, AMMs and MDMs were cultured with and without FLS for six hours after which cell surface marker expression and cytokine production were analyzed by flow cytometry and a bead-based multiplex assay, respectively. Results showed that regardless of exposure conditions, AMMs had significantly higher surface expression of CD163 and CD206 than MDMs. Incubation with FLS induced secretion of IL-1ß, IL-8, TNF-α and IFN-γ in AMMs, and IL-8, IL-10 and TNF-α in MDMs. These results suggest that AMMs have a greater proinflammatory response to in vitro FLS stimulation than MDMs, inferring differing roles in equine lung inflammation. Variability in recruitment and function of monocyte-macrophage populations warrant more detailed in vivo investigation in both homeostatic and diseased states.

Experience With Recombinant Human Bone Morphogenetic Protein-2 in Posterior Lumbar Interbody Fusion: A Retrospective Review of 1019 Procedures.

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with an activated collagen scaffold (Infuse; Medtronic, MN) has been used to facilitate lumbar intervertebral fusion; however, data regarding its efficacy are inconsistent. We aimed to assess the efficacy of rhBMP-2 when used in posterior lumbar interbody fusion (PLIF) by analyzing the rate of reoperation for nonunion and patient-reported outcome measures in a large retrospective case series. We also aimed to assess the impact of patient and surgical factors on rates of reoperation and determine frequency of complications. METHODS: Prospectively collected data from a single-surgeon database of consecutive PLIFs (minimum 18-month follow-up) were retrospectively analyzed. PLIF was performed with pedicle screw instrumentation, intervertebral spacers, and locally harvested bone graft to which rhBMP-2 and bone marrow aspirate (BMA) were added. Multivariate logistic regression was used to determine the influence of patient and surgical factors on the primary outcome: reoperation for confirmed nonunion. RESULTS: A total of 1019 operations at 1485 levels across 908 patients were analyzed. Mean duration of follow-up was 51.7 ± 30.0 months (range 18-172). Twelve patients required reoperation for nonunion (1.2%). Increasing body mass index was found to be significant in predicting reoperation (OR 1.114, P = 0.046). Postoperative radiculitis was common (42%) but transient in most cases. There were significant and sustained improvements in patient-reported outcome measures postoperatively. Four cases of osteolysis and 5 of epidural cyst were recorded, and a reduction of rhBMP-2 dose seemed to ameliorate these sequelae. CONCLUSION: In this large retrospective observational study, PLIF performed with rhBMP-2 and BMA resulted in a low rate of clinically significant nonunion and significant improvement in patient-reported outcomes. Transient radiculitis was common. Osteolysis and epidural cyst formation were rare and possibly related to dosage. CLINICAL RELEVANCE: rhBMP-2 is effective when used in PLIF, resulting in a high rate of fusion and improved patient outcomes, and it has an acceptable safety profile.

Zoonotic Ancylostoma ceylanicum Hookworm Infections, Ecuador.

Ancylostoma ceylanicum hookworms are zoonotic parasites that can infect humans. To detect autochthonous transmission, we analyzed human fecal samples collected in 2000. Multiparallel quantitative PCR detected infection in persons who had never traveled outside Ecuador. These data indicate human transmission of A. ceylanicum in the Americas, although endemicity remains unknown.

Effects of equine SALSA on neutrophil phagocytosis and macrophage cytokine production.

Salivary scavenger and agglutinin (SALSA) is a secreted protein with various immunomodulatory roles. In humans, the protein agglutinates and inactivates microorganisms, and inhibits the release of pro-inflammatory cytokines. Saliva, which is rich in SALSA, accelerates bacterial phagocytosis, but SALSA's contribution is unclear. In horses, the functions of SALSA in inflammation remain undetermined, so they were investigated through phagocytosis and cytokine assays. Equine SALSA was purified from duodenal tissue, which contains abundant SALSA. To assess phagocytosis, fluorescently-labelled bacteria were incubated with 20, 10, 5, or 2.5 µg/mL of SALSA or phosphate buffered saline (PBS), and then incubated at 37°C or on ice with whole blood from seven healthy horses. Fluorescence was measured by gating on neutrophils using a flow cytometer, and compared between groups. To assess effects on cytokine production, alveolar macrophages were isolated from bronchoalveolar lavage fluid of five healthy horses and cultured in serum-free media for 24 hours with different concentrations of SALSA plus 1 µg/mL lipopolysaccharide (LPS), only LPS, or only media. Cytokines were measured in supernatant using an equine-specific multiplex bead immunoassay. There was significantly greater phagocytosis in samples incubated at 37°C compared to incubation on ice. Samples incubated with 20 µg/mL of SALSA at 37°C had less phagocytosis compared to samples with 10 or 2.5 µg/mL SALSA, or PBS. Alveolar macrophages incubated with SALSA plus LPS released significantly less CXC motif chemokine ligand 1, interleukin-8, interleukin-10, and tumor necrosis factor α, and more granulocyte colony stimulating factor (G-CSF), compared to macrophages incubated with LPS alone. These findings indicate anti-inflammatory effects, which may be due to interference with toll-like receptor 4 recognition of LPS or downstream signaling. Increase in G-CSF following incubation with SALSA suggests a novel mechanism for immunoregulation of alveolar macrophages by SALSA, addressing a knowledge gap regarding its functions in horses.

Strongy Detect: Preliminary Validation of a Prototype Recombinant Ss-NIE/Ss-IR Based ELISA to Detect Strongyloides stercoralis Infection.

BACKGROUND: Strongyloides stercoralis (Ss) is the etiological agent of strongyloidiasis, a neglected tropical disease of global concern. Laboratory diagnosis of strongyloidiasis is most often based on detection of antibodies against antigens in an enzyme linked immunosorbent assay (ELISA). Herein, we report a preliminary validation study of newly developed IgG4- and/or IgG- based ELISAs to detect strongyloidiasis (Strongy Detect, InBios) incorporating a cocktail of 2 previously described recombinant antigens, Ss-NIE and Ss-IR. METHODS: The sensitivity and specificity were determined by using the assay in 150 cryopreserved serum samples from humans known to be Ss infected (n = 74), helminth uninfected (n = 47), or infected with a helminth other than Ss [n = 29). The treatment associated dynamics of antibody detection were then assessed using 35 paired samples obtained before and after definitive therapy. RESULTS: The IgG and IgG4 assays were 99% and 96% sensitive, respectively, and 99% and 100% specific, respectively. Neither the IgG or IgG4 assay showed cross reactions with sera from those infected with other helminths. Although ELISA values did decline post-treatment few returned to levels below the cutoff for infection. CONCLUSION: Strongy Detect is the most sensitive and specific commercialized immunoassay for detection of strongyloidiasis. The assay remains positive for greater than a year post-treatment.

Validation of a Novel Ultrasound Simulation Model for Teaching Foundation-Level Ultrasonography Skills to Veterinary Students.

Veterinary ultrasonography is a complex, advanced skill requiring repetitive exposure and supervision to gain competence. Consequently, newly graduated veterinarians are underprepared and lack the resources to achieve basic ultrasound proficiency upon graduation. Ultrasound simulation has been proposed as an adjunct educational tool for teaching entry-level ultrasound skills to student veterinarians. The objectives of this multicentric prospective observational cohort study were to describe the development of a novel ultrasound training model, establish model construct and face validity, and seek participant feedback. The model was constructed using three-dimensional silicone shapes embedded in ballistics gel within a glass container. A novice cohort of 15 veterinary students and 14 expert participants were prospectively enrolled in the study. Each cohort underwent training and assessment phases using a simulation model. Participants were asked to (a) determine shape location, (b) identify shape type using a shape bank, and (c) measure shape axes using the caliper tool. Time for each phase was recorded. Anonymous post-participation survey feedback was obtained. For most shapes (4/6), experts performed significantly better than novices in identifying shape type and location. Generally, no significant difference was found in mean axis shape measurements between cohorts or compared to the true mean axis measurements. No significant difference was found in scan time for either phase. This study's results support the validation of this ultrasound simulation model and may demonstrate early evidence for its use as a training tool in the veterinary curriculum to teach entry-level ultrasound skills.

Bronchial brush cytology, endobronchial biopsy, and SALSA immunohistochemistry in severe equine asthma.

Horses with severe equine asthma (SEA), also known as heaves and recurrent airway obstruction, have persistent neutrophilic inflammation of the lower airways. Cytologic evaluation of bronchoalveolar lavage (BAL) fluid is commonly used to confirm the clinical diagnosis of SEA. However, the utility of microscopic assessment of bronchial brushings, endobronchial biopsies, and immunohistochemical detection of disease-associated biomarkers for the diagnosis of SEA remain poorly characterized. Salivary scavenger and agglutinin (SALSA) has anti-inflammatory properties and downregulated gene expression in SEA; therefore, it was investigated as a tissue biomarker for airway and systemic inflammation. Six asthmatic and 6 non-asthmatic horses were exposed to an inhaled challenge. Before and after challenge, samples of BAL fluid, bronchial brushing, and endobronchial biopsy were collected. Location of SALSA in biopsies was determined, and immunohistochemical label intensity was computed using image analysis software. Serum amyloid A (SAA) was measured to assess systemic inflammation. After challenge, neutrophil proportions were significantly higher in asthmatic versus non-asthmatic horses in BAL fluid (least squares means, 95% confidence interval: 80.9%, 57.2% to 93.1%, vs 3.6%, 1.1% to 10.7%) and in brush cytology slides (39.5%, 7.7% to 83.6%, vs 0.2%, 0% to 2.3%), illustrating the potential of brush cytology as an alternate modality to BAL for assessing intraluminal inflammation. Bronchial histopathologic findings and intensity of SALSA immunolabeling in surface and glandular epithelium were similar in asthmatic and non-asthmatic horses, indicating limited changes in bronchial tissue from the inhaled challenge. Increases in SAA indicated systemic inflammation, but SALSA immunolabeling did not change significantly.

Factors Associated with Fatality in Ontario Thoroughbred Racehorses: 2003-2015.

Ontario's Alcohol and Gaming Commission records equine racing fatalities through its Equine Health Program. The present study examined all Thoroughbred fatalities from 2003 to 2015, inclusive, to identify associations. Official records and details of fatalities were combined in multivariable logistic regression modelling of 236,386 race work-events (433 fatalities), and 459,013 workout work-events (252 fatalities). Fatality rates were 2.94/1000 race starts (all fatalities) and 1.96/1000 (breakdowns only) with an overall rate of 2.61% or 26.1 fatalities/1000 horses. Comparison with published reports reveals rates to be high. Musculoskeletal injury was the predominant complaint and there was a high incidence of horses dying suddenly. Liability was high for young horses early in the season with a differential according to sex and whether a male horse was gelded. Horses undertaking repeated workouts had a higher liability and liability was higher in workouts for horses switching from dirt/synthetic to turf racing and for young horses in sprints. Race distance was not significant but high fatality rates in some large field, distance races combined with effects of age and workload identified groups at particular risk. As field size increased, fatality liability increased for early-finishing horses. Findings suggest jockey strategy could be an important factor influencing fatalities. Probability of fatality declined over the study period. Findings indicate that rapid accumulation of workload in animals early in their preparation is likely to be damaging. Fatality fell toward the end of a season and for horses with a long career history of successful performance; however, horses not exhibiting this robustness and staying power represent the population of greatest concern. Associations may be characterised as representing sources of stress, current or cumulative, and identifying at-risk animals on this basis may be as productive as targeting specific, discrete mechanisms suspected to contribute to individual fatalities.

Effect of anesthesia duration on the quality of recovery in horses undergoing elective and emergency surgeries using the same anesthetic protocol.

The objective of this study was to investigate the effect of anesthesia duration on the quality of recovery in horses. The medical records of horses that were anesthetized and underwent surgery for elective and emergency soft tissue and orthopedic conditions from 2013 to 2019 were reviewed. Horses included in the study (N = 305) fulfilled the following requirements: all had the same premedication/induction protocol and the same balanced anesthesia for maintenance and were anesthetized by the same, experienced Board-certified anesthesiologist. A standardized anesthetic recovery score was completed for all horses to evaluate their recovery and the following interactions were assessed: age, body weight, breed, sex, American Society of Anesthesiologists status, type of surgical procedure, occurrence of hypotension, use of dobutamine, number of additional doses of xylazine/ketamine after isoflurane discontinuation, anesthesia duration, post-anesthetic sedation, and end-tidal isoflurane concentration during maintenance and at the time of transfer to the recovery room. These interactions were assessed based on the quality of recovery score using logistic regression. Duration of anesthesia (P = 0.021) and age (P = 0.003) negatively affected the quality of recovery. The odds of a worse recovery score were increased by 1.20-fold (1.03, 1.41; lower and upper limits) for every additional 30 min of anesthesia duration, while the odds of a worse recovery score were increased by 1.09-fold (1.03, 1.16) for every additional 1 y of age. In conclusion, the results of this retrospective study indicate that increasing the anesthesia duration negatively affects the quality of recovery in horses undergoing routine and emergency surgical procedures.

L'objectif de cette étude était d'étudier l'effet de la durée de l'anesthésie sur la qualité de la récupération chez les chevaux. Les dossiers médicaux des chevaux qui ont été anesthésiés et ont subi une intervention chirurgicale élective et d'urgence des tissus mous et orthopédique de 2013 à 2019 ont été examinés. Les chevaux inclus dans l'étude (N = 305) remplissaient les conditions suivantes : tous avaient le même protocole de prémédication/d'induction et la même anesthésie équilibrée pour l'entretien et ont été anesthésiés par le même anesthésiste expérimenté et certifié par le Board. Un score de récupération anesthésique standardisé a été réalisé pour tous les chevaux afin d'évaluer leur récupération et les interactions suivantes ont été évaluées : âge, poids corporel, race, sexe, statut American Society of Anesthesiologists, type d'intervention chirurgicale, survenue d'hypotension, utilisation de dobutamine, nombre de doses supplémentaires de xylazine/kétamine après l'arrêt de l'isoflurane, la durée de l'anesthésie, la sédation post-anesthésique et la concentration d'isoflurane en fin d'expiration pendant l'entretien et au moment du transfert en salle de réveil. Ces interactions ont été évaluées sur la base de la qualité du score de récupération en utilisant la régression logistique. La durée de l'anesthésie (P = 0,021) et l'âge (P = 0,003) ont affecté négativement la qualité de la récupération. Les probabilités d'un pire score de récupération ont été multipliées par 1,20 (1,03, 1,41; limites inférieure et supérieure) pour chaque 30 min supplémentaire de durée d'anesthésie, tandis que les chances d'un pire score de récupération ont été multipliées par 1,09 (1,03, 1,16) pour chaque année d'âge supplémentaire. En conclusion, les résultats de cette étude rétrospective indiquent que l'augmentation de la durée de l'anesthésie affecte négativement la qualité de la récupération chez les chevaux subissant des interventions chirurgicales de routine et d'urgence.(Traduit par Docteur Serge Messier).

RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNA.

Angiostrongylus cantonensis is one of the leading causes of eosinophilic meningitis worldwide. A field-deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA), was developed to target a region predicted to be highly repeated in the A. cantonensis genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using A. cantonensis genomic DNA, the limit of detection was found to be 1 fg/µl by both fluorometer measurement and visual reading. All clinical samples known to be positive for A. cantonensis from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e., Toxocara sp. and Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35°C and 40°C. The assay successfully detected 1 fg/µl of A. cantonensis genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point-of-contact molecular assay capable of sensitively detecting A. cantonensis by producing visually interpretable results with minimal instrumentation.

Factors Associated with Mortality in Ontario Standardbred Racing: 2003-2015.

Factors associated with mortality in standardbred racehorses were assessed through a retrospective annualized cohort study of all-cause mortality from 2003-2015 (n = 978) (identified in the Ontario Racehorse Death Registry). Race and qualifying data for official work-events were also gathered (1,778,330 work-events, 125,200 horse years). Multivariable logistic regression analysis revealed sex, age, and indices of workload and intensity and their interactions to be strongly associated with mortality. Track class, race versus qualifying performance, and work-event outcome (finish position, scratched, or failed to finish) also influenced mortality odds, which increased as performance slowed. Intense competition at higher performance levels and qualifying races at lower levels carried particularly high odds. Though occurring frequently, musculoskeletal injury was less frequent than all other presenting problems combined. Industry structure contributes to mortality through interaction between horse characteristics and the competition environment. This substrate may be amenable to management to minimize liability, but incident-specific triggers may represent chance factors and be relatively difficult to identify or control. Differentiating between substrate and trigger when studying specific clinical problems may provide greater clarity and yield in identifying underlying causes. Mortality may reflect a continuum of circumstances, cumulative impacts of which might be identified before a fatal event occurs.

Risk of adjacent segment disease after 'topping-off' multi-level lumbar fusions with posterior dynamic stabilisers: an observational cohort study.

PURPOSE: To determine whether 'topping-off' lumbar fusions, using posterior dynamic stabilising devices (PDSs) with specific biomechanical parameters, reduces the risk of adjacent segment disease (ASD). METHODS: Survival analysis of two non-randomised cohorts, with or without 'topping-off' (T/O or NoT/O), compared the risk of further surgery for ASD following multi-level posterior lumbar interbody fusion (PLIF). The study sample comprised consecutive patients, aged 55 + years, with degenerative pathology at 2, 3 or 4 levels. The NoT/O cohort underwent surgery between August 1993 and September 2019 (n = 425) and the T/O cohort between September 2011 and September 2019 (n = 146). Comparison of ASD risk between cohorts used Cox proportional hazards (CPH) modelling and Kaplan-Meier survivorship analysis. RESULTS: Analysis was completed on 571 operations across 507 patients. Median follow-up was 63 months (range 0.3-196) and 37 months (range 1.7-98) for the NoT/O and T/O cohorts, respectively. Of 423 patients, 125 (29.6%) patients in the NoT/O cohort underwent further surgery for ASD and 16/145 (11.03%) in the T/O cohort. The hazard ratio (T/O: NoT/O) from the CPH model was 0.42 (95% CL: 0.24-0.74, P = 0.003). Mean annual incidence across the first 5 years was 5.0% in the NoT/O cohort compared with 2.8% in the T/O cohort (P = 0.029). No patient required surgery or developed ASD at a 'topped-off' level. Two patients developed asymptomatic pedicle screw loosening at the level of the PDS device. PROMs were similar between cohorts. CONCLUSION: This large, non-randomised, observational study found an approximately 60% reduction in further surgery for ASD with the use of the PDS to 'top-off' PLIF fusions. PDS device-related complications were very low.

Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010-2020) and results for a novel, highly sensitive qPCR assay.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells µL-1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.

AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections.

BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Correlation of subclinical gastrointestinal nematode parasitism with growth and reproductive performance in ewe lambs in Ontario.

Infection with gastrointestinal nematode parasites (GINs) is an important cause of productivity loss on sheep farms in Ontario and worldwide. However, efforts to quantify the effect of GIN infection on growth have demonstrated mixed results. Furthermore, there has been limited investigation of their effect on reproductive performance. This study evaluated the effect of subclinical GIN parasitism on growth and reproductive performance of ewe lambs under Ontario grazing conditions. Rideau cross ewe lambs (n = 140) born in spring 2016 on a farm in central Ontario were followed for two years from before weaning through to November 2017, including their first lambing and lactation. These animals grazed from May to November of each year and were sampled every 6-8 weeks during both grazing seasons and once at mid-gestation in March 2017. At each sampling the ewe lambs were weighed, body condition scores assigned, fecal egg counts (FECs) performed, and pasture samples collected to assess number of infective GIN larvae. Study animals with a FEC of 500 eggs per gram or higher were selectively treated with anthelmintics to prevent morbidity and mortality. Fecal samples were cultured to determine infecting GIN species, and climate data were obtained from a weather station 26 km away from the farm. Precipitation levels and numbers of infective larvae on pasture were low during the first grazing season but were more typical of Ontario conditions in the second grazing season. The three most common GIN species were Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus spp. General linear mixed models were generated for weight change over time, litter size at lambing, and weaning weights of offspring. Despite moderate peak GIN burdens in both grazing seasons, FEC was not significantly associated with weight change or litter size, apart from periparturient egg rise in study ewe lambs with larger litters (p = 0.05). Significant positive quadratic and negative linear associations were identified between late lactation FECs and offspring weaning weights; the association between FECs and weaning weights changed from negative to positive at a FEC of 361 eggs per gram. These results indicate that when GIN burdens are moderate as evidenced by fecal egg counts and infection is subclinical, there appears to be low to no impact on growth and reproductive performance in ewe lambs in the first 18 months of life. This suggests that when GIN parasitism is regularly monitored and controlled using targeted selective treatment, animal performance is minimally affected.

Correlation of salivary antibody to carbohydrate larval antigen (CarLA) with health and gastrointestinal nematode parasitism in sheep under Ontario grazing conditions.

Leveraging mucosal immunity is a promising method for controlling gastrointestinal nematode (GIN) parasitism in sheep. Salivary antibody to carbohydrate larval antigen (sCarLA), a heritable measure of immunity to third-stage GIN larvae (L3), has been successfully applied to genetic improvement programs in New Zealand. However, sCarLA levels wane in the absence of ongoing GIN exposure. New Zealand's temperate climate permits year-round exposure to L3, but cold winters in boreal regions such as Ontario, Canada interrupt exposure for five months or more. This study investigated associations between sCarLA levels, GIN parasitism, and indicators of overall health in sheep grazing under Ontario conditions. A commercial flock of 140 Rideau cross ewe lambs were followed from approximately 30 days of age in May 2016 until November 2017, including lambing and lactation in the spring of 2017. Every 6-8 weeks during the grazing season and at mid-gestation in March 2017, fecal egg counts were performed, blood collected to assess serum albumin, globulin, and hematocrit, and pasture samples obtained to confirm exposure to infective larvae. Measurements of sCarLA level were performed at the beginning, middle, and end of each grazing season, and at mid-gestation. Spearman's rank correlation coefficients were calculated to compare sCarLA levels over time, and general linear mixed models created to evaluate associations between sCarLA levels, GIN fecal egg count, hematocrit, serum albumin, and serum globulin. Levels of sCarLA followed a similar seasonal pattern to GIN fecal egg counts with a 6-8 week delay; much higher sCarLA levels were observed in the second grazing season. The proportion of the flock with detectable sCarLA (≥ 0.3 units/mL) was 68.3 % by the end of the first grazing season, declined over winter to 43.9 % at lambing, and approached 100 % after 3 months of grazing in the second grazing season. Correlations between sCarLA levels over time were consistently positive, of weak to moderate strength, and significant (p < 0.05). At all time points, sCarLA level was significantly (p < 0.001) and negatively associated with fecal egg counts. The flock displayed minimal variability in hematocrit, serum albumin, and serum globulin; none of which were significantly associated with sCarLA levels. These results suggest that sCarLA can be maintained over winter and is a useful measure of immunity to GINs in sheep under Ontario grazing conditions.