Sucrose permeability as a marker of gastric mucosal integrity in the horse : Feasibility, assay development and field validation of a blood test for diagnosis of gastric ulcers in horses

Gastric ulcers can develop in foals and horses of all breeds and uses, and the term equine gastric ulcer syndrome (EGUS) has been coined to describe this disease because of its multifactorial and complicated nature. Currently, detection of EGUS by gastroscopy is the only reliable ante mortem method for definitive diagnosis in horses, and is considered the gold standard against which all other diagnostic tests are compared. Disadvantages of gastroscopy are that it requires the horse to be sedated, it is not readily available to most veterinarians, it is an inefficient expenditure of time, and requires a minimum level of expertise to perform and interpret. A urine sucrose test has been reported to be a reliable method of detecting gastric ulcers in horses; however, technical difficulties associated with urine collection have limited the practical value of the test. It was hypothesized that blood sucrose concentration following nasogastric administration of sucrose can be used as a simple, economical alternative to reliably and practically detect gastric ulcers in horses; and a series of studies were subsequently conducted to develop and validate the test, including determination of the feasibility of the method; sucrose assay development and standardization; and field validation through determination of the performance characteristics of the test in selected populations of horses. The feasibility of the method was determined in 12 adult horses with naturally occurring gastric ulceration. Horses with moderate to severe gastric ulceration demonstrated a significant increase in serum sucrose concentrations at 30, 45, 60 and 90 minutes following nasogastric administration of sucrose. Peak sucrose concentrations occurred at 45 minutes and were correlated with ulcer severity. It was concluded that the determination of sucrose concentration in blood is a feasible alternative to urine when performing sucrose permeability testing in the horse, and may represent a useful screening test for identifying horses with endoscopically visible gastric ulceration. An accurate, yet practical and cost-effective method for quantifying sucrose in equine serum that can be applied to sucrose permeability testing in the horse was subsequently developed and validated using gas chromatography with flame ionization detection (GC-FID). The assay provided an acceptable degree of linearity, accuracy and precision at concentrations of sucrose as low as 2.34 µmol/L and as high as 20.45 µmol/L. Percentage recovery of sucrose from serum ranged from 89 – 102%; and repeatability and intermediate precision (RSD %) ranged from 3.6 to 6.7 % and 4.1 to 9.3 % respectively. The limit of detection was 0.73 µmol/L. It was concluded that the method is valid; and can be applied to the assessment of gastric permeability in the horse. The performance characteristics of the test were subsequently assessed in a large group of adult horses and foals with naturally occurring gastric ulceration by comparing it to gastroscopy as the gold standard. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL); glandular lesions (GDL); squamous lesions (SQL); and clinically significant lesions (CSL) at 45 and 90 minutes after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using receiver operator characteristics (ROC) curves and calculating the area under the curve (AUC). For each lesion type, sucrose concentration in blood was compared to gastroscopy as the gold standard; and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Ulcer grading was performed blindly by one observer; and the results were validated by comparing them with that of two other observers, and calculating the level of agreement. Cut-off values were selected manually to optimize Se. Because of concerns over the validity of the gold standard, additional Se, Sp, and lesion prevalence data were estimated and compared using Bayesian latent class analysis. Using the traditional gold standard approach, the prevalence of GL; GDL; SQL and CSL for adult horses was 83%; 70%; 53% and 58% respectively. For foals, the prevalence of GL; GDL; SQL and CSL before weaning was 21%; 9%; 7% and 8% respectively; and increased to 98%; 59%; 97% and 82% respectively after weaning. At the selected cut-offs, Se ranged from 51% to 79% for adult horses; and 84% to 95% for foals, depending upon the lesion type and time of sampling. Sp was poor, ranging from 43% to 72%; and 47% to 71% in adult horses and foals respectively. Estimates of Se and Sp were consistently higher in foals when using a Bayesian approach, however there was little difference between the methods when compared in adult horses. It was concluded that blood sucrose is neither a sensitive or specific test for detecting EGUS in adult horses and is therefore unsuitable as a screening test this study population. In contrast, blood sucrose appears to be a sensitive test for detecting EGUS in foals. Due to its poor specificity, it is not expected that the sucrose blood test will replace gastroscopy, however it may represent a clinically useful screening test to identify foals that may benefit from gastroscopy. Bayesian latent class analysis may represent an alternative method to evaluate the diagnostic accuracy of gastric permeability tests in an attempt to avoid bias associated with the assumption that gastroscopy is a perfect test.Mahahaavoja esiintyy sekä varsoilla että aikuisilla hevosilla rotuun tai käyttöön katsomatta. Useiden taustatekijöiden ja monimuotoisen taudinkuvan vuoksi sairaudesta käytetään termiä EGUS eli Equine Gastric Ulcer Syndrome. Mahalaukun tähystys eli gastroskopia on diagnostiikan ideaalisin eli gold standard -menetelmä ja ainoa luotettava kliininen diagnosointitapa. Tähystys vaatii hevosen rauhoittamisen, eikä kaikilla eläinlääkäreillä ole toimenpiteeseen vaadittavaa välineistöä eikä tulkintaan vaadittavaa kokemusta. Virtsan sokerin (sakkaroosin eli sukroosin) analyysin on raportoitu olevan luotettava menetelmä hevosen mahahaavan diagnostiikassa, mutta virtsan kerääminen on teknisesti hankalaa eikä testi näin ollen ole käytännöllinen. Tutkimushypoteesina oli että sokerin nenänieluletkulla tapahtuvan annostelun jälkeen veren sokerikonsentraation analysointi olisi yksinkertainen, käytännöllinen ja taloudellinen vaihtoehto todentaa hevosen mahahaavat luotettavasti. Testin kehittämiseksi ja validoimiseksi tehtiin sarja tutkimuksia: sokerin mittaustestin kehittäminen ja standardisointi, testaustekniikan käytännöllisyys, sekä testin validointi valikoidussa hevospopulaatiossa. Metodin soveltuvuus määritettiin 12 aikuisella hevosella, joilla oli todettu mahahaava. Hevosilla, joilla oli kohtalainen tai vakava mahahaava, sokerin konsentraatio nousi merkittävästi 30, 45, 60 and 90 minuuttia sokerin nenänieluletkutuksen jälkeen. Sokerin huippupitoisuus oli 45 minuutin kohdalla, ja korreloi haavauman vakavuuden kanssa. Johtopäätöksenä todettiin, että veren sokerin läpäisevyystesti verrattuna virtsan sokerin konsentraation mittaamiseen on käyttökelpoinen vaihtoehto seulontatestinä jolla löydetään mahahaavahevoset. Sokerin mittaamiseen hevosen seerumista kehitettiin ja validoitiin tarkka, käytännöllinen ja taloudellinen menetelmä käyttäen kaasukromatografiaa ja liekki-ionisaatio detektoria (GC-FID). Analyysi oli riittävän tarkka ja lineaarinen sekä matalilla (2.34 µmol/L) että korkeilla (20.45 µmol/L) sokerikonsentraatioilla. Seerumiin sokeripitoisuuden määrä vaihteli välillä 89 - 102%; toistettavuus välillä 3.6 - 6.7%, sekä tarkkuus (intermediate precision; RSD %) välillä 4.1 - 9.3%. Menelmän mittausraja oli 0.73 µmol/L. Menetelmän todettiin olevan pätevä ja sitä pystyttiin käyttämään hevosen mahalaukun läpäisevyyden tutkimisessa. Testin toimivuutta mahalaukun tähystykseen verrattuna arvioitiin laajan aineiston avulla. Ryhmissä oli sekä aikuisia hevosia että varsoja. Veren sakkaroosipitoisuus arvioitiin 45 ja 90 minuuttia 1 g/kg sokeriannoksen nenänieluletkuksen jälkeen. Pitoisuuden diagnostinen tarkkuus mahalaukun (GL), ja sen eri osien (rauhasalue (GDL), keratinisoitunut alue (SQL)) vaurioiden, sekä kliinisesti merkittävien vaurioiden (CSL) arvioitiin käyttäen ROC- käyrää ja laskemalla alle jäävä ala (AUC). Sokerin pitoisuutta veressä eri vauriotyypeissä verrattiin gastroskopialöydöksiin (gold standard), herkkyys ja tarkkuus. Mahahaavojen luokittelun suoritti sokkoutettu tutkija, tulokset validoitiin kahden muun arvioinnilla ja näistä laskettiin yksimielisyyden taso (the level of agreement). Herkkyyden parantamiseksi raja-arvot valittiin manuaalisesti. Gastroskopia gold standard -testinä ei ole tarkka, joten herkkyys, tarkkuus ja vaurioiden esiintyvyys arvioitiin ja vertailtiin Bayesian latentilla luokka-analyysilla. Perinteisellä gold standard -menetelmällä vaurioiden esiintyvyydet aikuisella hevosella olivat GL 83%, GDL70%, SQL 53% ja CSL 58%. Varsoilla vaurioiden esiintyvyydet olivat GL 21%, GDL 9%, SQL 7% ja CSL 8% ennen vieroitusta, sen jälkeen ne nousivat 98%, 59%; 97% and 82%. Valituilla raja-arvoilla aikuisten hevosten testin herkkyys oli 51 - 79% ja varsojen 84-95% riippuen vaurion tyypistä ja näytteenottoajasta. Tarkkuus oli heikko vaihdellen 43-72% aikuisilla ja 47–71% varsoilla. Herkkyys ja tarkkuusarviot olivat kauttaaltaan korkeampia varsoilla, jos käytettiin Bayesian mallia, aikuisilla metodeilla ei ollut juuri eroa. Loppupäätelmänä todettiin että veren sakkaroosipitoisuuden testi ei ole herkkä tai tarkka testi hevosen mahahaavalle aikuisella hevosella, eikä sovellu seulontatestiksi. Sen sijaan varsoilla veren sokeripitoisuus näyttäisi olevan herkkä testi mahahaavan (EGUS) toteamiseen. Todennäköisesti testi ei korvaa tähystystä huonon spesifisyytensä takia, mutta se voisi toimia käytännöllisenä testinä arvioitaessa varsojen tarvetta tähystykseen. Bayesian latentti luokka- analyysi voi olla toimiva testivaihtoehto mahan läpäisevyyden arvioimiseen,,koska gold standard –testissä on puutteita

Diagnostic accuracy of blood sucrose as a screening test for equine gastric ulcer syndrome (EGUS) in adult horses

Background: Equine gastric ulcer syndrome (EGUS) is common in adult horses, particularly those involved in performance disciplines. Currently, detection of EGUS by gastroscopy is the only reliable ante mortem method for definitive diagnosis; however it is unsuitable as a screening test because it is expensive, time consuming, and is not readily available to most veterinarians. Sucrose permeability testing represents a simple, economical alternative to gastroscopy for screening purposes, and the feasibility of this approach in the horse has been previously reported. The aim of this study was to determine the diagnostic accuracy of blood sucrose as a screening test for EGUS in a large group of adult horses with and without naturally occurring gastric disease. Results: One hundred and one adult horses with or without naturally occurring gastric ulceration were studied. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL), glandular lesions (GDL), squamous lesions (SQL), and clinically significant lesions (CSL) at 45 and 90 min after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using receiver operator characteristics (ROC) curves and calculating the area under the curve (AUC). For each lesion type, sucrose concentration in blood was compared to gastroscopy, as the gold standard, and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Ulcer grading was performed blindly by one observer; and the results were validated by comparing them with that of two other observers, and calculating the level of agreement. Cut-off values were selected manually to optimize Se. The prevalence of GL, GDL, SQL, and CSL was 83, 70, 53 and 58% respectively. At the selected cut-offs, Se ranged from 51 to 79% and Sp ranged from 43 to 72%, depending upon the lesion type and time of sampling. Conclusions: Blood sucrose is neither a sensitive or specific test for detecting EGUS in this population of adult horses with naturally occurring gastric ulceration. Further studies aimed at evaluating the performance characteristics of the test in different study populations are warranted. Given the limitations of endoscopy, due consideration should also be given to alternative methods for comparison of blood sucrose with a gold standard.Peer reviewe

Diagnostic accuracy of blood sucrose as a screening test for equine gastric ulcer syndrome (EGUS) in adult horses

Background: Equine gastric ulcer syndrome (EGUS) is common in adult horses, particularly those involved in performance disciplines. Currently, detection of EGUS by gastroscopy is the only reliable ante mortem method for definitive diagnosis; however it is unsuitable as a screening test because it is expensive, time consuming, and is not readily available to most veterinarians. Sucrose permeability testing represents a simple, economical alternative to gastroscopy for screening purposes, and the feasibility of this approach in the horse has been previously reported. The aim of this study was to determine the diagnostic accuracy of blood sucrose as a screening test for EGUS in a large group of adult horses with and without naturally occurring gastric disease. Results: One hundred and one adult horses with or without naturally occurring gastric ulceration were studied. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL), glandular lesions (GDL), squamous lesions (SQL), and clinically significant lesions (CSL) at 45 and 90 min after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using receiver operator characteristics (ROC) curves and calculating the area under the curve (AUC). For each lesion type, sucrose concentration in blood was compared to gastroscopy, as the gold standard, and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Ulcer grading was performed blindly by one observer; and the results were validated by comparing them with that of two other observers, and calculating the level of agreement. Cut-off values were selected manually to optimize Se. The prevalence of GL, GDL, SQL, and CSL was 83, 70, 53 and 58% respectively. At the selected cut-offs, Se ranged from 51 to 79% and Sp ranged from 43 to 72%, depending upon the lesion type and time of sampling. Conclusions: Blood sucrose is neither a sensitive or specific test for detecting EGUS in this population of adult horses with naturally occurring gastric ulceration. Further studies aimed at evaluating the performance characteristics of the test in different study populations are warranted. Given the limitations of endoscopy, due consideration should also be given to alternative methods for comparison of blood sucrose with a gold standard.Peer reviewe

Ileal hypertrophy and associated true diverticulum as a cause of colic in a horse

A 4-year-old Thoroughbred gelding underwent an explorative celiotomy for a suspected small intestinal obstruction. During surgery, an impacted diverticulum of the ileum was suspected, necessitating a jejunocaecostomy. The owner opted for euthanasia. On post-mortem examination and histopathology, a true diverticulum on the mesenteric side of the ileum, with ileal hypertrophy, was diagnosed.http://www.jsava.co.zaam2017Companion Animal Clinical StudiesParaclinical Science

Pharmacokinetics of an intravenous and oral dose of enrofloxacin in white rhinoceros (Ceratotherium simum)

South Africa currently loses over 1000 white rhinoceros (Ceratotherium simum) each year to poaching incidents, and numbers of severely injured victims found alive have increased dramatically. However, little is known about the antimicrobial treatment of wounds in rhinoceros. This study explores the applicability of enrofloxacin for rhinoceros through the use of pharmacokinetic‐pharmacodynamic modelling. The pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were evaluated in five white rhinoceros after intravenous (i.v.) and after successive i.v. and oral administration of 12.5 mg/kg enrofloxacin. After i.v. administration, the half‐life, area under the curve (AUCtot), clearance and the volume of distribution were 12.41 ± 2.62 hr, 64.5 ± 14.44 μg ml−1 hr−1, 0.19 ± 0.04 L h−1 kg−1, and 2.09 ± 0.48 L/kg, respectively. Ciprofloxacin reached 26.42 ± 0.05% of the enrofloxacin plasma concentration. After combined i.v. and oral enrofloxacin administration oral bioavailability was 33.30 ± 38.33%. After i.v. enrofloxacin administration, the efficacy marker AUC24: MIC exceeded the recommended ratio of 125 against bacteria with an MIC of 0.5 μg/mL. Subsequent intravenous and oral enrofloxacin administration resulted in a low Cmax: MIC ratio of 3.1. The results suggest that intravenous administration of injectable enrofloxacin could be a useful drug with bactericidal properties in rhinoceros. However, the maintenance of the drug plasma concentration at a bactericidal level through additional per os administration of 10% oral solution of enrofloxacin indicated for the use in chickens, turkeys and rabbits does not seem feasible.The German Academic Exchange Service (DAAD), the University of Pretoria, the South African Veterinary Association (SAVA) and Bayer Animal Health.http://wileyonlinelibrary.com/journal/jvp2020-05-01hj2019Companion Animal Clinical StudiesParaclinical Science

Diagnostic accuracy of blood sucrose as a screening test for equine gastric ulcer syndrome (EGUS) in weanling foals

Background: Equine gastric ulcer syndrome is an important cause of morbidity in weanling foals. Many foals are asymptomatic, and the development of an inexpensive screening test to ensure an early diagnosis is desirable. The objective of this study was to determine the diagnostic accuracy of blood sucrose for diagnosis of EGUS in weanling foals. Results: 45 foals were studied 7 days before and 14 days after weaning. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL); glandular lesions (GDL); squamous lesions (SQL) and clinically significant gastric lesions (CSL) at 45 and 90 min after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using ROC curves and calculating the AUC. For each lesion type, sucrose concentration in blood was compared to gastroscopy; and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Cut- off values were selected manually to optimize Se. Because of concerns over the validity of the gold standard, additional Se, Sp, and lesion prevalence data were subsequently estimated and compared using Bayesian latent class analysis. Using the frequentist approach, the prevalence of GL; GDL; SQL and CSL before weaning was 21; 9; 7 and 8% respectively; and increased to 98; 59; 97 and 82% respectively after weaning. At the selected cut- off, Se ranged from 84 to 95% and Sp ranged from 47 to 71%, depending upon the lesion type and time of sampling. In comparison, estimates of Se and Sp were consistently higher when using a Bayesian approach, with Se ranging from 81 to 97%; and Sp ranging from 77 to 97%, depending upon the lesion type and time of sampling. Conclusions: Blood sucrose is a sensitive test for detecting EGUS in weanling foals. Due to its poor specificity, it is not expected that the sucrose blood test will replace gastroscopy, however it may represent a clinically useful screening test to identify foals that may benefit from gastroscopy. Bayesian latent class analysis represents an alternative method to evaluate the diagnostic accuracy of the blood sucrose test in an attempt to avoid bias associated with the assumption that gastroscopy is a perfect test.Peer reviewe

Maturation of the functional mouse CRES amyloid from globular form

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms

Towards a new phenotype for tick resistance in beef and dairy cattle:a review

About 80% of the world's cattle are affected by ticks and tick-borne diseases, both of which cause significant production losses. Cattle host resistance to ticks is the most important factor affecting the economics of tick control, but it is largely neglected in tick-control programs due to technical difficulties and costs associated with identifying individual-animal variation in resistance. The present paper reviews the scientific literature to identify factors affecting resistance of cattle to ticks and the biological mechanisms of host tick resistance, to develop alternative phenotype(s) for tick resistance. If new cost-effective phenotype(s) can be developed and validated, then tick resistance of cattle could be genetically improved using genomic selection, and incorporated into breeding objectives to simultaneously improve cattle productive attributes and tick resistance. The phenotype(s) could also be used to improve tick control by using cattle management. On the basis of the present review, it is recommended that three possible phenotypes (haemolytic analysis measures of skin hypersensitivity reactions simplified artificial tick infestations) be further developed to determine their practical feasibility for consistently, cost-effectively and reliably measuring cattle tick resistance in thousands of individual animals in commercial and smallholder farmer herds in tropical and subtropical areas globally. During evaluation of these potential new phenotypes, additional measurements should be included to determine the possibility of developing a volatile-based resistance phenotype, to simultaneously improve cattle resistance to both ticks and biting flies. Because the current measurements of volatile chemistry do not satisfy the requirements of a simple, cost-effective phenotype for use in commercial cattle herds, consideration should also be given to inclusion of potentially simpler measures to enable indirect genetic selection for volatile-based resistance to ticks