Genomic association and further characterisation of faecal immunoglobulin A deficiency in German Shepherd dogs.

BACKGROUND: Immunoglobulin A (IgA) deficiency, chronic enteropathies and exocrine pancreatic insufficiency (EPI) have a high prevalence in German Shepherd dogs (GSD). This prospective study determined the prevalence of faecal IgA deficiency (IgAD) in GSD and investigated several candidate genes and the canine genome for a region or locus co-segregating with IgAD in GSD. Faecal IgA concentrations were quantified and genomic DNA was extracted from 8 GSD with an undetectable faecal IgA (classified as IgAD) and 80 non-IgAD GSD. The canine minimal screening set II microsatellite markers were genotyped, with evidence of an association at p < 1.0 × 10-3 . Faecal IgA concentrations were also tested for an association with patient clinical and biochemical variables. RESULTS: Allele frequencies observed using the candidate gene approach were not associated with faecal IgAD in GSD. In the genome-wide association study (GWAS), the microsatellite marker FH2361 on canine chromosome 33 approached statistical significance for a link with IgAD in GSD (p = 1.2 × 10-3 ). A subsequent GWAS in 11 GSD with EPI and 80 control GSD revealed a significant association between EPI and FH2361 (p = 8.2 × 10-4 ). CONCLUSIONS: The lack of an association with the phenotype of faecal IgAD in GSD using the candidate gene approach and GWAS might suggests that faecal IgAD in GSD is a relative or transient state of deficiency. However, the prevalence of faecal IgAD in GSD appears to be low (<3%). The relationship between faecal IgAD, EPI and loci close to FH2361 on canine chromosome 33 in GSD warrants further investigation.

Histopathological findings and canine pancreatic lipase immunoreactivity in normal dogs and dogs with inflammatory and neoplastic diseases of the pancreas.

BACKGROUND: Diagnosis of pancreatic diseases in dogs is still challenging because of variable clinical signs, which do not always correspond with clinical pathology and histopathological findings. OBJECTIVES: To characterize inflammatory and neoplastic pancreatic diseases of dogs and to correlate these findings with clinical findings and canine pancreatic lipase immunoreactivity (cPLI) results. ANIMALS: Tissue specimens and corresponding blood samples from 72 dogs submitted for routine diagnostic testing. METHODS: Four groups were defined histologically: (1) normal pancreas (n = 40), (2) mild pancreatitis (n = 8), (3) moderate or severe pancreatitis (acute, n = 11; chronic, n = 1), and (4) pancreatic neoplasms (n = 12). An in-house cPLI ELISA (<180 µg/L, normal; >310 µg/L, pancreatitis) was performed. RESULTS: In dogs with normal pancreas, 92.5% of serum cPLI results were within the reference range and significantly lower than in dogs with mild acute pancreatitis, moderate or severe acute pancreatitis and pancreatic tumors. In dogs with moderate or severe acute pancreatitis, cPLI sensitivity was 90.9% (95% confidence interval [CI], 58.7%-99.8%). Most dogs (9/12) with pancreatic tumors (group 4) had additional pancreatic inflammation and cPLI results were increased in 10 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: High cPLI indicates serious acute pancreatitis but underlying pancreatic neoplasms should also be taken into consideration. This study confirms the relevance of histopathology in the diagnostic evaluation of pancreatic diseases.

Maternal adrenocorticotropin, cortisol, and thyroid hormone responses to all three-trimester equivalent repeated binge alcohol exposure: ovine model.

Alcohol-mediated alterations in hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid axis function are two proposed mechanisms by which alcohol causes neurodevelopmental injury to the fetus. We previously reported that third-trimester equivalent only alcohol exposure in sheep results in increases in the maternal and fetal adrenocorticotropin and cortisol levels, and decreases in the fetal thyroid hormones T(3) and T(4) and maternal T(3) levels. In this study, we wished to characterize the maternal HPA and hypothalamic-pituitary-thyroid hormone responses to repeated binge alcohol exposure during all three-trimester equivalents of pregnancy in sheep. Pregnant ewes received intravenous infusions of alcohol at doses of 0.75, 1.25, or 1.75 g/kg over 1h with mean peak blood alcohol concentrations of 90, 126, or 183 mg/dl, respectively, on 3 consecutive days each week beginning on gestational day (GD) 4. Maternal blood samples were collected on GDs 6, 40, 90, and 132. Maternal plasma concentrations of adrenocorticotropin and cortisol increased in response to the high alcohol dose, and the magnitude of these elevations was not different across gestation. Thyroid hormone levels were not different when comparing among treatment groups at any time point during gestation. However, there was an ontogenetic decrease in the maternal T(3) concentration beginning between GDs 6 and 40 and a decrease in maternal T(4) and free T(4) beginning between GDs 40 and 90. The current findings suggest that (1) maternal alcohol consumption at any time during gestation stimulates the HPA axis, (2) maternal HPA responsiveness to alcohol does not change across gestation, (3) binge alcohol exposure at these doses lasting all three-trimester equivalent of human brain development does not reduce maternal thyroid hormone concentration, (4) alterations in fetal thyroid function in response to alcohol exposure do not occur as a result of diminished maternal thyroid hormone contribution, and (5) there is an ontogenetic decrease in ovine maternal thyroid hormones over gestation.

Development of a fecal sample collection strategy for extraction and quantification of fecal immunoglobulin A in dogs.

OBJECTIVE: To develop a fecal sample collection strategy and quantification method for measurement of fecal IgA concentrations in dogs. SAMPLE POPULATION: Fecal samples from 23 healthy pet dogs of various breeds. PROCEDURES: Immunoglobulin A was extracted from fecal samples. An ELISA for the measurement of fecal IgA concentrations was established and analytically validated. Intraindividual variation of fecal IgA was determined by calculation of coefficients of variation. A sample collection strategy was developed on the basis of results of intraindividual variation of fecal IgA concentrations. A reference range for fecal IgA concentrations was determined. RESULTS: The method for extraction and quantification of fecal IgA was determined to be sufficiently sensitive, reproducible, accurate, and precise. On the basis of the intraindividual variability of our results, the determined fecal sample collection strategy required analysis of a total of 4 fecal samples/dog, with each fecal sample collected on 2 consecutive days with 28 days between sample collection periods (ie, days 1 and 2 followed by days 28 and 29). Reference range values for fecal IgA concentration were 0.22 to 3.24 mg/g of feces. CONCLUSIONS AND CLINICAL RELEVANCE: Methods of fecal IgA extraction and quantification used in our study allow for identification of dogs with consistently low fecal IgA concentrations. Use of these techniques will enable future investigations into possible associations between low fecal IgA concentrations and signs of gastrointestinal disease in dogs.

Purification and partial characterization of feline pepsinogen.

OBJECTIVE: To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species. SAMPLE POPULATION: Stomachs of 6 cats. PROCEDURE: A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition. RESULTS: Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10-fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum. CONCLUSIONS AND CLINICAL RELEVANCE: PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats.

The role of cortisol in chronic binge alcohol-induced cerebellar injury: Ovine model.

Women who drink alcohol during pregnancy are at high risk of giving birth to children with neurodevelopmental disorders. Previous reports from our laboratory have shown that third trimester equivalent binge alcohol exposure at a dose of 1.75 g/kg/day results in significant fetal cerebellar Purkinje cell loss in fetal sheep and that both maternal and fetal adrenocorticotropin (ACTH) and cortisol levels are elevated in response to alcohol treatment. In this study, we hypothesized that repeated elevations in cortisol from chronic binge alcohol are responsible at least in part for fetal neuronal deficits. Animals were divided into four treatment groups: normal control, pair-fed saline control, alcohol and cortisol. The magnitude of elevation in cortisol in response to alcohol was mimicked in the cortisol group by infusing pregnant ewes with hydrocortisone for 6 h on each day of the experiment, and administering saline during the first hour in lieu of alcohol. The experiment was conducted on three consecutive days followed by four days without treatment beginning on gestational day (GD) 109 until GD 132. Peak maternal blood alcohol concentration in the alcohol group was 239 ± 7 mg/dl. The fetal brains were collected and processed for stereological cell counting on GD 133. The estimated total number of fetal cerebellar Purkinje cells, the reference volume and the Purkinje cell density were not altered in response to glucocorticoid infusion in the absence of alcohol. These results suggest that glucocorticoids independently during the third trimester equivalent may not produce fetal cerebellar Purkinje cell loss. However, the elevations in cortisol along with other changes induced by alcohol could together lead to brain injury seen in the fetal alcohol spectrum disorders.