Automated Spectrophotometric Assays for the Measurement of Ammonia and Bicarbonate in Saliva of Horses: Analytical Validation and Changes in Equine Gastric Ulcer Syndrome (EGUS).

Ammonia (NH3) and bicarbonate (HCO3) have been related to gastric ulcers in humans. Ammonia is considered a possible cause of gastric ulcers, whereas bicarbonate has a protective function. The presence of ulcers in the stomach of horses is defined as Equine Gastric Ulcer Syndrome (EGUS), which is a frequent disease in this species, and it has been associated with changes in saliva composition, such as in analytes related to inflammation, immune system and oxidative stress. The objectives of this study were (1) to perform an analytical validation of two automated spectrophotometric assays, one for ammonia and one for bicarbonate, in the horses' saliva and (2) to evaluate their possible variations with EGUS. Analytical validation of the automated assays for ammonia and bicarbonate in the saliva of horses showed that both assays were precise and accurate. In addition, significantly higher values of ammonia and lower values of bicarbonate were found in the saliva of horses with EGUS compared to healthy horses. It can be concluded that ammonia and bicarbonate can be measured in the saliva of horses and that ammonia increases and bicarbonate decreases in this sample type could be related to the presence of EGUS in this species.

Changes in the saliva proteome analysed by gel-proteomics in horses diagnosed with equine gastric ulcer syndrome (EGUS) at diagnosis and after successful treatment.

Equine gastric ulcer syndrome (EGUS) is currently one of the more frequent diseases in horses. We aimed to identify changes in the salivary proteome in horses with EGUS at diagnosis and after successful treatment by using gel proteomics. Saliva samples were collected from nine horses with EGUS before and after treatment and nine matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. Horses with EGUS had increases in proteins such as adenosine deaminase (ADA), triosephosphate isomerase, keratins and immuno-globulin heavy constant mu and decreases in carbonic anhydrase (CA), albumin and prolactin-induced protein. These changes would indicate various physiopathological mechanisms involved in this disease, such as the activation of the immune system, decreased stomach defence mechanisms and inflammation. The treated horses presented lower expression levels of thioredoxin (TRX) after a successful treatment, in proteomics analysis and also measured with a commercially available ELISA kit. Overall, horses with EGUS have protein changes in their saliva when measured with gel proteomics compared with healthy horses, and they also showed changes after successful treatment. These proteins could be potential biomarkers for detection and monitoring treatment response in EGUS.

Carnot Cycles in a Harmonically Confined Ultracold Gas across Bose-Einstein Condensation.

Carnot cycles of samples of harmonically confined ultracold 87Rb fluids, near and across Bose-Einstein condensation (BEC), are analyzed. This is achieved through the experimental determination of the corresponding equation of state in terms of the appropriate global thermodynamics for non-uniform confined fluids. We focus our attention on the efficiency of the Carnot engine when the cycle occurs for temperatures either above or below the critical temperature and when BEC is crossed during the cycle. The measurement of the cycle efficiency reveals a perfect agreement with the theoretical prediction (1-TL/TH), with TH and TL serving as the temperatures of the hot and cold heat exchange reservoirs. Other cycles are also considered for comparison.

Changes in Proteins in Saliva and Serum in Equine Gastric Ulcer Syndrome Using a Proteomic Approach.

Changes in the salivary proteome in 12 horses with the two diseases included in equine gastric ulcer syndrome (EGUS), equine glandular gastric disease (EGGD) (n = 6) and equine squamous gastric disease (ESGD) (n = 6), were evaluated using a high-resolution LC-MS/MS analysis of TMT-labelled peptides and compared to 10 healthy control horses. Serum was also analysed for comparative purposes. The comparison between the horses with EGGD and controls showed significant changes in 10 salivary proteins, whereas 36 salivary proteins were differently abundant between ESGD and control groups. The most upregulated proteins in the case of EGGD were related to immune activation whereas, in horses with ESGD, the most significantly changed proteins were associated with squamous cell regulation and growth. Compared to serum, saliva showed a higher number of proteins with significant changes and a different pattern of changes. The proteins identified in our study, in addition to providing new information about the pathophysiological mechanisms in these diseases, could have the potential to be novel biomarkers for the diagnosis or monitoring of EGGD and ESGD.

Changes in Oxidative Status Biomarkers in Saliva and Serum in the Equine Gastric Ulcer Syndrome and Colic of Intestinal Aetiology: A Pilot Study.

Changes in the oxidative status of the blood of horses suffering from gastric ulcers and colic of intestinal aetiology (CIE) have been reported. However, saliva can also be a source of biomarkers of oxidative status. Therefore, this study aims to validate automated assays for the measurement of oxidative status biomarkers (ferric reducing ability of saliva/serum-FRAS/FRAP, cupric reducing antioxidant capacity-CUPRAC, the Trolox equivalent antioxidant capacity-TEAC, uric acid, and advanced oxidation protein products-AOPP) in the saliva and serum of horses, to assess their changes in the different ulcer gastric diseases (squamous-ESGD and glandular-EGGD) and CIE, and to evaluate their relationship with serum amyloid A (SAA), adenosine deaminase (ADA), and the systemic inflammatory response syndrome (SIRS) status. The assays showed a low imprecision and good linearity with enough sensitivity in both fluids. In EGGD, higher levels of FRAS, uric acid, and AOPP in saliva were observed compared to the healthy group, correlating with the salivary ADA levels. Horses with CIE showed increases in uric acid concentrations in serum associated with their SIRS status and outcome of the disease. In conclusion, analytes related to the oxidative status can be measured in the saliva and serum from horses by automated assays, and some of them can potentially be assessed as biomarkers in horses with gastric ulcers and CIE.

Endoscopic Endocautery Polypectomy for the Treatment of Duodenal and Gastric Polyps in a Cat.

Endoscopic polypectomy is commonly performed in human medicine, with large-scale studies reported. However, few reports have described its use in veterinary medicine and, specifically, the procedure in the case of duodenal polyps in cats has not been reported. A 7 kg 14-year-old cat presented with recurrent vomiting for several months. Gastroduodenoscopy revealed a pedunculated polyp at the pyloric antrum and another in the duodenum, with its head protruding into the pylorus. Endoscopic polypectomy was performed using an electrosurgical snare with no recurrence of clinical signs after six months. Duodenal polypectomy in cats may be difficult because of space limitation but it can be safe, minimally invasive, and successful, thus avoiding more invasive surgical techniques. Endoscopic polypectomy may be a viable alternative to surgery in cats with gastric and duodenal polyps.

Pharmacokinetics of Metformin in Combination With Sitagliptin in Adult Horses After Enteral Administration.

Insulin dysregulation (ID) is a common metabolic disorder in horses. Recently, incretin hormone release has been suggested to be involved in ID in horses. In human medicine, metformin and sitagliptin are commonly used in combination for metabolic syndrome. This combination could be useful in treating ID in horses. However, no pharmacokinetics data have been reported in this species. The objective of the present study was to establish the plasma concentration-time profile and to derive pharmacokinetics data for a combination of metformin and sitagliptin in horses after enteral administration. Six healthy adult Purebred Spanish horses were used. A metformin (15 mg/kg) plus sitagliptin (1.5 mg/kg) preparation was administered by intragastric route (IG) as an enteral solution. Blood samples were collected from 0 to 48 hours after IG drug administration. Plasma concentrations of metformin and sitagliptin were measured using high performance liquid chromatography methods. The t½λz for metformin was 2.9 hours and for sitagliptin 21 hours. The Cmax was 442 ± 84 mg/L within 0.9 hours for metformin and 94 ± 14 mg/L within 1.3 hours for sitagliptin. No adverse effects were observed, and the combination of metformin and sitagliptin was well tolerated. Therefore, these results suggest that metformin plus sitagliptin might be a combination to consider in horses with ID. Additional studies are needed to establish the effectiveness and tolerance in equids affected by endocrine disorders.

BENIGN IDIOPATHIC ESOPHAGEAL STRICTURE IN A LION ( PANTHERA LEO): DILATION BY AN ACHALASIA BALLOON.

A 1-yr old female lion ( Panthera leo) was referred with a 10-mo history of dysphagia for solid food (meat), episodic regurgitation, and poor weight gain. Esophagoscopy confirmed an esophagitis (midesophagus) and a stricture estimated to be of 13 mm diameter. This was subsequently dilated using a 20-mm-diameter balloon for 2 min followed by a 35-mm achalasia balloon for 3 min. The etiology remains undetermined in spite of a thorough history. The animal progressed satisfactorily, reaching 124 kg after 1 yr and has had no further signs. To the authors' knowledge, neither idiopathic esophageal stricture nor dilation using an achalasia balloon has been reported in Panthera spp.

An Atypical Case of Leishmaniasis Associated with Chronic Duodenitis in a Dog.

We describe an atypical case of duodenal leishmaniasis in a boxer dog presenting with chronic diarrhea and poor general condition. Antidiarrheic therapy was previously administered without success and inflammatory bowel disease localized to the small intestine was suspected, given the chronic clinical signs and by ruling out other known causes of gastrointestinal inflammation. Endoscopic biopsy of duodenum showed a moderate increase in lamina propria lymphocytes, plasma cells, and macrophages. Basophilic bodies were seen in the cytoplasm of numerous macrophages, suggestive of Leishmania spp, confirmed by immunostaining, and a diagnosis of granulomatous duodenitis associated to Leishmania infection was made. After 7 mo of therapy, a significant clinical improvement and weight gain were observed, and endoscopic histology showed no evidence of Leishmania. A progressive decline of anti-leishmanial antibody titer was also observed during follow-up. This report emphasizes the importance of atypical symptoms and the unusual location of visceral leishmaniasis, suggesting the need to consider leishmaniasis in the differential diagnosis of canine chronic enteritis, especially in endemic areas.

Effect of diet/atorvastatin on atherosclerotic lesions associated to nonalcoholic fatty liver disease in chickens.

Comparative histological examination of both liver and the supra-aortic arteries have not previously examined the consequences of atherosclerosis and nonalcoholic fatty liver disease (NAFLD), and their response to diet and atorvastatin therapy. This study evaluates the effects of diet alone or in combination with atorvastatin therapy on the progression/regression of atherosclerosis and its correlation with NAFLD. This research was performed on a cohort of chickens on standard (SD) or hyperlipidemic diets (HD), either with or without atorvastatin therapy. The development of atherosclerotic lesions was assessed by histology, immunohistochemistry and quantitative image analysis and correlated with liver histology. The lowest levels of atherosclerotic lesions were found in animals on the HD for 3 months, followed by 3 months of SD in combination with oral atorvastatin. There was a strong association between the histologic findings of atherosclerosis and those of NAFLD. These studies show that standard diet and atorvastatin therapy can positively affect both arterial and hepatic lesions, influencing the regression of the changes. These results support the hypothesis that NAFLD and atherosclerosis may be actually two aspects of a shared disease and suggest the possibility of regression of both disorders with dietary and pharmacologic manipulations.

Characterization of anal double balloon endoscopy in the dog.

Retrograde (anal) exploration of the canine small intestine by conventional endoscopy is restricted to the ileum, but double balloon endoscopy (DBE) has recently been validated using the oral approach. In this study, anal DBE was performed on nine healthy dogs to characterize the efficiency, exploration dynamics and safety of the technique. DBE was successful in all dogs; the average estimated insertion depth of the endoscope was about 2m and took approximately 1h to achieve. No complications or related adverse clinical effects were observed, and pancreatitis did not occur following the procedure. Anal DBE is viable and safe in the dog within the limits of the study, and has the potential to facilitate the diagnosis and treatment of gastrointestinal diseases, not only in portions of the colon and ileum, but also in portions of the distal jejunum.

Cortisol, adrenocorticotropic hormone, serotonin, adrenaline and noradrenaline serum concentrations in relation to disease and stress in the horse.

No detailed comparative data are available on the hormonal parameters of horses suffering from a number of diseases. The aim of our study was to measure concentrations of cortisol, adrenocorticotropic hormone (ACTH), serotonin, adrenaline and noradrenaline in horses with various diseases and following surgery, to assess the response of the HPA axis and adrenal medulla. Blood samples were obtained from six groups of horses comprising a total of 119 animals as follows: laminitis, acute abdominal syndrome (AAS), castration surgery, acute diseases, chronic diseases and healthy controls. Serum hormonal concentrations were determined for each group for comparison. Statistically significant differences between all groups and controls were found for cortisol, ACTH (except for castration), serotonin and adrenaline concentrations but only in horses with laminitis and AAS for noradrenaline. No statistically significant differences were found between males and females. The largest changes in the pituitary-adrenal axis activity occurred mainly in acute diseases, laminitis and in the AAS group.

A case of lymphocytic-plasmacytic jejunitis diagnosed by double-balloon enteroscopy in a dog.

A 3 yr old male English setter dog was presented for evaluation of a 6-wk history of intermittent diarrhea. After standard gastroduodenoscopy and colonoscopy showed normal mucosa, double-balloon endoscopy (DBE) was used via both oral and anal approaches. Gross changes consistent with inflammation in the jejunum were seen, and biopsy specimens were obtained. Histologic analysis confirmed a diagnosis of lymphocytic-plasmacytic jejunitis. Clinical remission of the disease occurred after 3 mo of therapy with prednisone, metronidazole, and a novel protein diet. Use of DBE has not been previously reported in dogs with inflammatory bowel disease, and isolated lymphocytic-plasmacytic jejunitis has not been described. The described cases of intestinal inflammatory disease diagnosed by conventional endoscopy were related to pathologic changes in the duodenum, ileum or colon, but not the jejunum. The main advantage of the DBE technique allowed examination of portions of the small intestine (jejunum) that were not commonly accessible by standard endoscopic techniques, and permitted a minimally invasive collection of biopsy samples compared with surgical biopsy. This case highlights the need to consider using DBE in animals with gastrointestinal disorders, whose symptoms are not readily explained by routine tests, conventional endoscopy, and dietary or therapeutic trials.

Assessment of liver steatosis in chicken by using acoustic radiation force impulse imaging: preliminary results.

OBJECTIVE: To evaluate acoustic radiation force impulse (ARFI) imaging as a non-invasive tool for quantification of the grades of liver steatosis in chickens. METHODS: We used two different diets: a standard diet (SD group) and a hyperlipidaemic diet (HD group). The ARFI technique was performed in all the animals in the right hepatic lobe and shear wave velocity (SWV) was measured and expressed in metres per second (m/s). Plasma lipid levels were analysed. Steatosis was quantified by using semiquantitative analysis. Statistical analysis was used and Pearson's correlation coefficient was calculated. RESULTS: Mean SWV was 0.94 ± 0.16 m/s (range 0.8-1.3 m/s) in the SD group and 1.91 ± 0.25 m/s (range 1.3-2.2 m/s) in the HD group (p < 0.001). The lowest SWVs (≤1.3 m/s) corresponded to the chickens in the SD group, with 100% of the animals returning a score of 0, whereas the range of SWV in the HD group chickens was between 1.6 and 2.2 m/s. A substantial correlation was observed between SWVs with histological semiquantitative analysis of steatosis (r = 0.85, p < 0.001). CONCLUSION: ARFI imaging is a non-invasive diagnostic tool that allows discrimination between the grades of liver steatosis in chickens.

Effect of atorvastatin and diet on non-alcoholic fatty liver disease activity score in hyperlipidemic chickens.

Non-alcoholic steatohepatitis (NASH) is part of the spectrum of non-alcoholic fatty liver disease (NAFLD), which includes from simple steatosis and steatohepatitis, to the most severe cirrhosis and carcinoma, which develops in the absence of excessive alcohol intake. NAFLD is the most common liver disorder in affluent societies. There is no proven treatment for NAFLD/NASH. One of the most frequent adverse effects of statins is an increase in hepatic aminotransferases. Studies that evaluate if the benefits of statins overcome the risks in NASH are lacking. The present study was conceived to explore the effect of both atorvastatin and diet on regression of steatohepatitis, using a chicken experimental model induced by a hyperlipidemic diet (HD). Plasma lipid levels, liver enzymes and hepatic histopathology, as well as image analysis were performed to determine changes in liver lipid deposits and inflammatory infiltration. Features of steatosis, cell-ballooning, and inflammation were scored to obtain the NAFLD activity score (NAS). A severe level of steatosis was found in animals fed on HD. Atorvastatin treated groups showed smaller size of lipid deposits and a lower level of inflammation than non-treated groups. Atorvastatin therapy induced a significant reduction of hepatocellular damage, even though in the animals which continuously received a hyperlipidemic diet. The combination of atorvastatin therapy and a standard diet produced the lowest decrease of NAS. Our results show that atorvastatin therapy not only decreased plasmatic levels of cholesterol and triglycerides, but also induced a reduction of liver steatosis, inflammation and hepatocellular damage, without increasing plasmatic aminotransferase levels.

Electrocardiographic parameters in the clinically healthy Zamorano-leones donkey.

Limited information exists regarding electrocardiographic parameters in clinically healthy donkeys. The study was carried out in 75 healthy adult animals (40 females and 35 males) using the Einthoven standard II and base-apex leads. The P wave showed usually a bifid shape deflection. The QRS complex of the donkeys appeared in several forms: QR and R were the most frequent in limb lead II, and QS and QR in the base-apex lead. Most T waves presented a simple negative configuration in lead II and biphasic shape in the base-apex one. Mean heart rate value was 52 beats per minute. The direction of the QRS vector in lead II had a mean value of 91.4 degrees. We observed a lack of detected arrhythmias. Statistically significant differences were observed between sexes for several parameters. The electrocardiogram of Zamorano-leones donkey differs in several duration, amplitude and morphologic parameters from that of several breeds of horses and donkeys. This fact justifies obtaining values for a specific breed against which to compare values for the same breed.

Postanesthetic brachial triceps myonecrosis in a Spanish-bred horse.

This report describes a case of postanesthetic brachial triceps myonecrosis affecting only the left forelimb of a horse. A fatal unilateral postanesthetic myonecrosis has not been previously reported in the horse. This article describes the factors in the horse's history, the anesthetic protocol, and the treatment that may have led to this condition.

Hyperlipidemic chicken as a model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is part of the spectrum of non-alcoholic fatty liver disease (NAFLD), currently the most common cause of abnormal liver tests. Given the difficulty of studying all the factors involved in it in human populations, studies in animal models might provide crucial insights in the pathogenesis of steatohepatitis. Several physiological features predispose birds to fat deposition in the liver. The present study was conceived to explore the possibilities of the chicken fed a cholesterol and fat enriched diet as a model for steatohepatitis. We used two different diets: a standard growing mash (control group) and a standard growing mash enriched with 2% cholesterol and 20% palm oil (hyperlipidemic group). We investigated the effect of feeding a cholesterol and fat enriched diet, on plasma lipid levels, liver enzymes and hepatic histopathology. Semiquantitative and quantitative assessment by image analysis was performed to determine changes in lipid deposits and inflammatory infiltration. Statistically significant increases were observed in all plasma lipid parameters, liver macroscopic features, fat deposits and cell-ballooning of hepatocytes between control and hyperlipidemic animals. Significant differences were also observed in the inflammatory infiltration parameters (number of foci, density, area and maximal diameter). Results show that diet-induced hypercholesterolemia and hypertriglyceridemia are associated with severe impairment of liver histology (fat accumulation, inflammation and cell-ballooning), reproducing histological features of human NAFLD. This model, which is easy and reproducible, offers economic and technical advantages. Furthermore, the reversibility of the pathologic changes makes it suitable for drug intervention studies of steatohepatitis.

Efecto de la atorvastatina sobre la progresión y regresión de los depósitos grasos renales en un modelo experimental / Effect of atorvastatin on progression/regression of renal lipid accumulation in an experimental model

Introducción. Hay evidencias de la relación entre la arteriosclerosis y la enfermedad renal progresiva. Sin embargo, son escasos los estudios en humanos o animales en que se utilicen estatinas para valorar sus efectos sobre la lesión renal. Objetivos. El objetivo de este trabajo es estudiar a escala histológica el efecto de la atorvastatina sobre los fenómenos de progresión y regresión de los depósitos grasos en el riñón en un modelo experimental. Material y método. Para la realización de los protocolos experimentales se utilizaron 100 pollos White-Leghorn, sometidos a una dieta hiperlipémica en la fase de inducción y a diversos protocolos de dieta normal/dieta hiperlipémica y tratamiento o no con atorvastatina en la fase de intervención. Se realizó la valoración de la grasa renal mediante técnicas histológicas y análisis de imagen. Resultados. En el grupo control sano no se observó la existencia de depósitos grasos, mientras que el grupo aterogénico presentó grandes depósitos grasos. El grupo de regresión e intervención con atorvastatina presentó una menor presencia de grasa, lo que supuso una diferencia significativa con todos los grupos. También en el caso de este grupo, se encontró que el diámetro de la acumulación grasa es significativamente menor respecto al resto de los grupos. Conclusiones. Nuestro modelo experimental es, para el estudio del riñón graso, un modelo homogéneo y de fácil manejo. La retirada de la dieta grasa y la administración simultánea de atorvastatina da lugar a los índices más bajos de depósitos grasos renales. La retirada por sí sola de la dieta no produce efectos tan marcados. Del estudio de diferentes parámetros se deduce que la atorvastatina acelera la regresión y frena la progresión de las acumulaciones grasas renales (AU)

Introduction. There is evidence of a relationship between atherosclerosis and progressive kidney disease. However, information on the role of statins on kidney disease in humans and animal models is lacking. Objectives. To analyze the effect of atorvastatin on progression/regression of renal lipid accumulation in an experimental model using histological analysis. Material and method. We used 100 White-Leghorn chickens fed with a hyperlipemic diet (induction stage), followed by an interventional stage, in which the animals were fed a normal or hyperlipemic diet and administered atorvastatin or placebo. Assessment of renal lipid accumulation was made by histologic and imaging analyses. Results. In the healthy control group, no intracellular renal fat deposits or accumulations were found. The atherogenic group showed substantial lipid accumulations. The intervention group with suspension of the hyperlipemic diet and atorvastatin administration showed significant differences with the remaining groups, presenting the lowest fat accumulation. This group also showed significantly lower fat accumulation diameter with respect to the remaining groups. Conclusions. Our experimental model was useful and suitable for the study of renal fat accumulation. Hyperlipemic diet suspension and simultaneous atorvastatin therapy led to the lowest renal fat accumulation. Hyperlipemic diet suspension alone produced less pronounced results. The parameters studied indicate that atorvastatin was effective in accelerating regression of renal fat accumulation and in decreasing its progression (AU)

Fatal brodifacoum poisoning in a pony.

Fatal brodifacoum poisoning in a pony is described; this condition has not previously been reported in ponies. Discussion of what factors in the pony's history and treatment may have predisposed to the severity and ultimate death is provided.