Real-Time PCR Differential Detection of Neorickettsia findlayensis and N. risticii in Cases of Potomac Horse Fever.

Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine. PHF is caused by Neorickettsia risticii, and the recently rediscovered and classified N. findlayensis. PHF diagnosis is currently accomplished using serology or nested PCR. However, both methods cannot distinguish the two Neorickettsia species that cause PHF. Further, the current N. risticii real-time PCR test fails to detect N. findlayensis. Thus, in this study, two Neorickettsia species-specific real-time PCR assays based on Neorickettsia ssa2 and a Neorickettsia genus-specific real-time PCR assay based on Neorickettsia 16S rRNA gene were developed. The ssa2 real-time PCR tests differentiated N. findlayensis from N. risticii in the field samples for which infection with either species had been verified using multiple other molecular tests and culture isolation, and the 16S rRNA gene real-time PCR detected both Neorickettsia species in the samples. These tests were applied to new field culture isolates from three Canadian provinces (Alberta, Quebec, Ontario) and Ohio as well as archival DNA samples from suspected PHF cases to estimate the prevalence of N. findlayensis in different geographic regions. The results suggest that N. findlayensis frequently causes PHF in horses in Alberta and Quebec. The development of these tests will allow rapid, sensitive, and specific diagnosis of horses presenting with clinical signs of PHF. These tests will also enable rapid and targeted treatment and help develop broad-spectrum vaccines for PHF.

Diagnostic evaluation of insulin and glucose dynamics in light-breed horses receiving dexamethasone.

Objective: Insulin dysregulation is a hallmark of equine metabolic syndrome (EMS) and increases the risk for development of laminitis. Accurate diagnosis of insulin dysregulation is crucial for implementation of preventative strategies in this population. The objective was to assess the effects of dexamethasone administration on insulin and glucose dynamics in light-breed horses and assess the agreement of various diagnostic tests for insulin dysregulation [basal [insulin] (BI), oral sugar test (OST), and combined glucose-insulin test (CGIT)]. Animal: Fourteen adult light-breed horses. Procedure: Prospective, experimental study to assess insulin and glucose dynamics by performing basal insulin, OST, and CGIT before (baseline) and post-dexamethasone administration (0.08 mg/kg, PO, q24h) for 7 d. Insulin and glucose dynamics were assessed by the BI, OST, CGIT, and insulin sensitivity proxy measurements (RISQI, QUICKI, FGIR, HOMA-IR, IG) at the baseline and post-dexamethasone time points. Results: The OST area under the insulin and glucose curves were increased following dexamethasone treatment (P < 0.001 and P < 0.01, respectively). Basal insulin, OST [insulin] at 60 min and CGIT [insulin] at 45 min were increased at the post-dexamethasone time point (P < 0.001, < 0.001, and < 0.01). Similarly, time spent in the positive glucose phase during the CGIT was longer at the post-dexamethasone time point (P < 0.001). The proxy measurements for insulin sensitivity (RISQI, QUICKI, FGIR) were decreased (P < 0.01) and the proxy measurements for insulin resistance (HOMA-IR) and ß-cell function (IG) were increased after dexamethasone administration (P < 0.01). More horses were classified with following dexamethasone administration, based on the diagnostic criteria for basal insulin (P = 0.03), OST (P = 0.01), and CGIT (P < 0.01). Kappa coefficients, measuring agreement between basal insulin, OST, and CGIT, showed none to moderate agreement at the baseline time point. Conclusion: Dexamethasone administration at 0.08 mg/kg, PO, q24h for 7 d worsened insulin dysregulation in adult light-breed horses based on findings of a basal insulin, OST, CGIT, and insulin sensitivity proxy measurements. There was none to moderate agreement between the basal insulin, OST, CGIT for the diagnosis of insulin dysregulation. Clinical relevance: Horses administered dexamethasone at a dose of 0.08 mg/kg, PO, q24h for 7 d should be considered insulin dysregulation and appropriate preventative strategies should be implemented. The variability of diagnostic performance of common tests for insulin dysregulation (basal insulin, OST, CGIT) may affect clinical decisions; therefore, performing multiple tests, including proxy measurements, may improve diagnostic accuracy of insulin dysregulation.

Objectif: La dysrégulation de l'insuline est une caractéristique du syndrome métabolique équin (EMS) et augmente le risque de développement de la fourbure. Un diagnostic précis de la dysrégulation de l'insuline est crucial pour la mise en oeuvre de stratégies préventives dans cette population. L'objectif était d'évaluer les effets de l'administration de dexaméthasone sur la dynamique de l'insuline et du glucose chez les chevaux de race légère et d'évaluer la concordance de divers tests de diagnostic pour le dérèglement de l'insuline [insuline basale] (BI), test de sucre oral (OST) et un test glucose-insuline combiné (CGIT). Animal: Quatorze chevaux adultes de race légère. Procédure: Étude prospective et expérimentale pour évaluer la dynamique de l'insuline et du glucose en effectuant l'insuline basale, l'OST et le CGIT avant (valeur de base) et après l'administration de dexaméthasone (0,08 mg/kg, PO, q24h) pendant 7 jours. La dynamique de l'insuline et du glucose a été évaluée par les mesures indirectes de BI, de l'OST, du CGIT et de la sensibilité à l'insuline (RISQI, QUICKI, FGIR, HOMA-IR, IG) aux points temporels de base et post-dexaméthasone. Résultats: La zone OST sous les courbes d'insuline et de glucose a augmenté après le traitement à la dexaméthasone (P < 0,001 et P < 0,01, respectivement). L'insuline basale, l'OST [insuline] à 60 minutes et le CGIT [insuline] à 45 minutes ont augmenté au point temporel post-dexaméthasone (P < 0,001, < 0,001 et < 0,01). De même, le temps passé dans la phase de glucose positif pendant le CGIT était plus long au moment post-dexaméthasone (P < 0,001). Les mesures indirectes de la sensibilité à l'insuline (RISQI, QUICKI, FGIR) ont diminué (P < 0,01) et les mesures indirectes de la résistance à l'insuline (HOMA-IR) et de la fonction des cellules ß (IG) ont augmenté après l'administration de dexaméthasone (P < 0,01). Plus de chevaux ont été classés avec l'administration suivante de dexaméthasone, sur la base des critères de diagnostic de l'insuline basale (P = 0,03), OST (P = 0,01) et CGIT (P < 0,01). Les coefficients Kappa, mesurant la concordance entre l'insuline basale, l'OST et le CGIT, ont montré une concordance nulle à modérée au point de référence. Conclusion: L'administration de dexaméthasone à 0,08 mg/kg, PO, toutes les 24 h pendant 7 jours a aggravé la dysrégulation de l'insuline chez les chevaux adultes de race légère d'après les résultats d'une insuline basale, d'OST, de CGIT et de mesures indirectes de la sensibilité à l'insuline. Il n'y avait aucun accord à modéré entre l'insuline basale, l'OST, le CGIT pour le diagnostic de dysrégulation de l'insuline. Pertinence clinique: Les chevaux ayant reçu de la dexaméthasone à une dose de 0,08 mg/kg, PO, q24h pendant 7 jours doivent être considérés comme ayant un dérèglement de l'insuline et des stratégies préventives appropriées doivent être mises en oeuvre. La variabilité des performances diagnostiques des tests courants de dysrégulation de l'insuline (insuline basale, OST, CGIT) peut affecter les décisions cliniques; par conséquent, la réalisation de plusieurs tests, y compris des mesures indirectes, peut améliorer la précision du diagnostic du dérèglement de l'insuline.(Traduit par Dr Serge Messier).

Development of a Formative Assessment Rubric for Peer Evaluation of Teaching (FARPET) and Pilot Use in Veterinary Online Teaching.

Peer evaluation of teaching (PET) serves an important role as a component of faculty development in the medical education field. With the emergence of COVID-19, the authors recognized the need for a flexible tool that could be used for a variety of lecture formats, including virtual instruction, and that could provide a framework for consistent and meaningful PET feedback. This teaching tip describes the creation and pilot use of a PET rubric, which includes six fixed core items (lesson structure, content organization, audiovisual facilitation, concept development, enthusiasm, and relevance) and items to be assessed separately for asynchronous lectures (cognitive engagement-asynchronous) and synchronous lectures (cognitive engagement-synchronous, discourse quality, collaborative learning, and check for understanding). The instrument packet comprises the rubric, instructions for use, definitions, and examples of each item, plus three training videos for users to compare with authors' consensus training scores; these serve as frame-of-reference training. The instrument was piloted among veterinary educators, and feedback was sought in a focus group setting. The instrument was well received, and training and use required a minimum time commitment. Inter-rater reliability within 1 Likert scale point (adjacent agreement) was assessed for each of the training videos, and consistency of scoring was demonstrated between focus group members using percent agreement (0.82, 0.85, 0.88) and between focus members and the authors' consensus training scores (all videos: 0.91). This instrument may serve as a helpful resource for institutions looking for a framework for PET. We intend to continually adjust the instrument in response to feedback from wider use.

Association of the neutrophil-lymphocyte ratio with outcome in sick hospitalized neonatal foals.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) in human medicine is an objective biomarker that reflects prognosis. The NLR as an independent biomarker to help predict nonsurvival in hospitalized neonatal foals has not been thoroughly interrogated. OBJECTIVES/HYPOTHESIS: Retrospectively evaluate if the NLR at admission is associated with nonsurvival in sick hospitalized foals <4 days old. We hypothesized that a lower NLR will be associated with nonsurvival. ANIMALS: One thousand one hundred ninety-six client-owned foals <4 days old of any breed and sex: 993 hospitalized foals and 203 healthy foals. METHODS: Retrospective multicenter study. Medical records of foals presenting to 3 equine referral hospitals were reviewed. Foals were included if they had complete CBCs, sepsis scores, and outcome data. The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. Data were analyzed by nonparametric methods and univariate analysis. RESULTS: Of the 993 sick hospitalized foals, 686 were sick nonseptic and 307 were septic. The median NLR was lower in sick hospitalized foals (median [95% confidence interval], 3.55 [0.5-13.9]) compared with healthy foals (6.61 [3.06-18.1]). Septic foals had the lowest NLR (2.00 [0.20-9.71]). The NLR was lower in nonsurviving (1.97 [1.67-2.45]) compared with surviving foals (4.10 [3.76-4.33]). Nonsurviving septic foals had the lowest NLR (1.47 [1.70-3.01]). Foals with a NLR of <3.06 or <1.6 at admission had odds ratio of 3.21 (2.24-4.29) and 4.03 (2.86-5.67) for nonsurvival, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: A NLR < 3.06 at admission in sick hospitalized foals is readily available and clinically useful variable to provide prognostic information.

Endothelial glycocalyx degradation in critically ill foals.

BACKGROUND: Endothelial glycocalyx (EG) degradation occurs in septic humans and EG products can be used as biomarkers of endothelial injury. Information about EG biomarkers and their association with disease severity is lacking in hospitalized foals. OBJECTIVES: Measure serum syndecan-1 (SDC-1), heparan sulfate (HS), angiopoietin-2 (ANG-2), aldosterone (ALD), and plasma atrial natriuretic peptide (ANP) concentrations and to determine their association with disease severity and death in hospitalized foals. ANIMALS: Ninety foals ≤3 days old. METHODS: Prospective, multicenter, longitudinal study. Foals were categorized into hospitalized (n = 74; 55 septic; 19 sick nonseptic) and 16 healthy foals. Serum ([SDC-1], [HS], [ANG-2], [ALD]) and plasma (ANP) were measured over 72 hours using immunoassays. RESULTS: Serum ([SDC-1], [HS], [ANG-2], [ALD]) and plasma (ANP) were significantly higher in hospitalized and septic than healthy foals (P < .05). Serum (ANG-2) and plasma (ANP) were significantly higher in hospitalized nonsurvivors than in survivors (P < .05). On admission, hospitalized foals with serum (HS) > 58.7 ng/mL had higher odds of nonsurvival (odds ratio [OR] = 6.1; 95% confidence interval [CI] = 1.02-36.7). Plasma (ANP) >11.5 pg/mL was associated with the likelihood of nonsurvival in hospitalized foals (OR = 7.2; 95% CI = 1.4-37.4; P < .05). Septic foals with serum (ANG-2) >1018 pg/mL on admission had higher odds of nonsurvival (OR = 6.5; 95% CI =1.2-36.6; P < .05). CONCLUSION AND CLINICAL IMPORTANCE: Critical illness in newborn foals is associated with EG degradation and injury, and these biomarkers are related to the severity of disease on admission and the outcome of sick foals.

Effect of 5'-adenosine monophosphate-activated protein kinase agonists on insulin and glucose dynamics in experimentally induced insulin dysregulation in horses.

BACKGROUND: 5'-adenosine monophosphate-activated protein kinase (AMPK) agonists, particularly resveratrol (RES), have not been extensively evaluated for their effect on insulin dysregulation (ID) in horses. OBJECTIVES: Evaluate the effects of treatment with RES (10 mg/kg PO q12h), metformin (MET; 30 mg/kg PO q12h), and aspirin (ASP; 20 mg/kg PO q24h) on experimentally induced ID. ANIMALS: Thirty-three healthy, adult, light-breed horses. METHODS: Unblinded, placebo-controlled, experimental trial evaluating effects of AMPK agonists (RES, MET, and ASP) on experimentally induced ID. Horses were randomly assigned to a treatment group (RES, MET/ASP, RES/ASP, RES/MET/ASP, or placebo [CON]) after induction of ID with dexamethasone (0.08 mg/kg PO q24h for 7 days). Frequently sampled insulin-modified IV glucose tolerance tests (FSIGTT) and oral sugar tests (OST) were performed at baseline, 7 days after ID, and ID plus 7 days of treatment. Minimal model and OST variables were compared between (1-way ANOVA) and within (1-way ANOVA for repeated measures) groups over time to determine effects of treatment on ID. RESULTS: Administration of dexamethasone for 14 days resulted in significantly altered insulin and glucose dynamics (SI, DI, basal [glucose], and [insulin]) and produced clinical signs of laminitis in 5 out of 33 (15%) of horses included in the study. Combination therapy with RES, MET, and ASP did not significantly improve insulin and glucose dynamics in horses with experimentally induced ID. CONCLUSIONS AND CLINICAL IMPORTANCE: Metabolic testing before glucocorticoid administration should be considered in horses with clinical signs of metabolic syndrome.

Energy endocrine physiology, pathophysiology, and nutrition of the foal.

Most homeostatic systems in the equine neonate should be functional during the transition from intra- to extrauterine life to ensure survival during this critical period. Endocrine maturation in the equine fetus occurs at different stages, with a majority taking place a few days prior to parturition and continuing after birth. Cortisol and thyroid hormones are good examples of endocrine and tissue interdependency. Cortisol promotes skeletal, respiratory, cardiovascular, thyroid gland, adrenomedullary, and pancreatic differentiation. Thyroid hormones are essential for cardiovascular, respiratory, neurologic, skeletal, adrenal, and pancreatic function. Hormonal imbalances at crucial stages of development or in response to disease can be detrimental to the newborn foal. Other endocrine factors, including growth hormone, glucagon, catecholamines, ghrelin, adipokines (adiponectin, leptin), and incretins, are equally important in energy homeostasis. This review provides information specific to nutrition and endocrine systems involved in energy homeostasis in foals, enhancing our understanding of equine neonatal physiology and pathophysiology and our ability to interpret clinical and laboratory findings, therefore improving therapies and prognosis.

Comparison of insulin sensitivity between healthy neonatal foals and horses using minimal model analysis.

The equine neonate is considered to have impaired glucose tolerance due to delayed maturation of the pancreatic endocrine system. Few studies have investigated insulin sensitivity in newborn foals using dynamic testing methods. The objective of this study was to assess insulin sensitivity by comparing the insulin-modified frequently sampled intravenous glucose tolerance test (I-FSIGTT) between neonatal foals and adult horses. This study was performed on healthy neonatal foals (n = 12), 24 to 60 hours of age, and horses (n = 8), 3 to 14 years of age using dextrose (300 mg/kg IV) and insulin (0.02 IU/kg IV). Insulin sensitivity (SI), acute insulin response to glucose (AIRg), glucose effectiveness (Sg), and disposition index (DI) were calculated using minimal model analysis. Proxy measurements were calculated using fasting insulin and glucose concentrations. Nonparametric statistical methods were used for analysis and reported as median and interquartile range (IQR). SI was significantly higher in foals (18.3 L·min-1· µIU-1 [13.4-28.4]) compared to horses (0.9 L·min-1· µIU-1 [0.5-1.1]); (p < 0.0001). DI was higher in foals (12 × 103 [8 × 103-14 × 103]) compared to horses (4 × 102 [2 × 102-7 × 102]); (p < 0.0001). AIRg and Sg were not different between foals and horses. The modified insulin to glucose ratio (MIRG) was lower in foals (1.72 µIUinsulin2/10·L·mgglucose [1.43-2.68]) compared to horses (3.91 µIU insulin2/10·L·mgglucose [2.57-7.89]); (p = 0.009). The homeostasis model assessment of beta cell function (HOMA-BC%) was higher in horses (78.4% [43-116]) compared to foals (23.2% [17.8-42.2]); (p = 0.0096). Our results suggest that healthy neonatal foals are insulin sensitive in the first days of life, which contradicts current literature regarding the equine neonate. Newborn foals may be more insulin sensitive immediately after birth as an evolutionary adaptation to conserve energy during the transition to extrauterine life.

Glucagon, insulin, adrenocorticotropic hormone, and cortisol in response to carbohydrates and fasting in healthy neonatal foals.

BACKGROUND: The endocrine pancreas and hypothalamic-pituitary-adrenal axis (HPAA) are central to energy homeostasis, but information on their dynamics in response to energy challenges in healthy newborn foals is lacking. OBJECTIVES: To evaluate glucagon, insulin, ACTH, and cortisol response to fasting and carbohydrate administration in healthy foals. ANIMALS: Twenty-two healthy Standardbred foals ≤4 days of age. METHODS: Foals were assigned to fasted (n = 6), IV glucose (IVGT; n = 5), PO glucose (OGT; n = 5), and PO lactose (OLT; n = 6) test groups. Blood samples were collected frequently for 210 minutes. Nursing was allowed from 180 to 210 minutes. Plasma glucagon, ACTH, serum insulin, and cortisol concentrations were measured using immunoassays. RESULTS: Plasma glucagon concentration decreased relative to baseline at 45, 90, and 180 minutes during the OLT (P = .03), but no differences occurred in other test groups. Nursing stimulated marked increases in plasma glucagon, serum insulin, and glucose concentrations in all test groups (P < .001). Plasma ACTH concentration increased relative to baseline at 180 minutes (P < .05) during fasting and OLT, but no differences occurred in other test groups. Serum cortisol concentration increased relative to baseline during OLT at 180 minutes (P = .04), but no differences occurred in other test groups. Nursing resulted in decreased plasma ACTH and serum cortisol concentrations in all test groups (P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: The endocrine response to enterally and parenterally administered carbohydrates, including the major endocrine response to nursing, suggests that factors in milk other than carbohydrates are strong stimulators (directly or indirectly) of the endocrine pancreas and HPAA.

Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells.

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.