Validation of serum gamma-glutamyl transferase activity and body weight information for identifying dairy calves that are too young to be transported to auction markets in Canada.

Dairy calves are at risk of being stressed when transported during the first week of life. A new Canadian federal rule will forbid transportation of calves younger than 9 d old to auction market. However, in the absence of reliable information to determine birth date, other indirect methods would be of interest. This study aimed to determine the prediction accuracy of body weight, Brix refractometry, and serum gamma-glutamyl transferase (GGT) activity for determining if a calf was not fit to be transported (i.e., <9 d old). For this purpose, we used 284 calves with a known birth date from a cross-sectional and a prospective cohort study. A logistic regression model was built based on multivariable analysis as well as a misclassification cost term analysis. Because of the collinearity between GGT activity and Brix value and lower discrimination of Brix value, the GGT activity was retained for the main model. The final logistic regression model contained body weight and log-transformed GGT activity value. The misclassifications of the logistic model was minimized using a model probability threshold ≥0.55 with a sensitivity of 70.4% and a specificity of 77.3%. This probability threshold was relatively robust for various prevalence and false negative to false positive cost ratios. The prediction accuracy of this model was moderate at the individual level, but is helpful in calves with a reasonable suspicion of being less than 9 d old.

Prenatal exposure to an environmentally relevant mixture of Canadian Arctic contaminants decreases male reproductive function in an aging rat model.

Elevated levels of organochlorines (OC) have been reported in Inuit populations in the Arctic. We hypothesized that prenatal exposure to a Canadian Arctic OC mixture adversely affects male reproductive function and health with age. Sprague-Dawley female rats (F0) were gavaged with an environmentally relevant concentration of an Arctic OC mixture or corn oil (Control) during mating with untreated males until parturition (F1 litters). After postnatal day (PND) 90, the weights of the OC F1 males differed dramatically relative to Controls (P<0.05; n=10) and they exhibited respiratory distress. Except for possible thinning of the alveolar barrier, histological observation of the lungs revealed no apparent pathology to explain the respiratory distress. At PND 365, OC F1 males had reduced relative reproductive organ weights and lower sperm quality than Controls (P<0.05). At PND 90, OC F1 males were subfertile (P<0.05), but were infertile at PND 365. In conclusion, environmentally relevant prenatal OC exposure reduced reproductive function and health in aging male rats, providing new insight into the effects of early-life exposures to these contaminants.

Surgical repair of propagating condylar fractures of the third metacarpal/metatarsal bones with cortical screws placed in lag fashion in 26 racehorses (2007-2015).

BACKGROUND: Despite the recommendation of plate fixation for propagating condylar fractures of the third metacarpal (McIII) or third metatarsal bone (MtIII), lag screw fixation can be a viable surgical option. OBJECTIVES: To evaluate short-term outcome and long-term racing performance of horses that underwent lag screw fixation of long condylar fractures of the McIII/MtIII. STUDY DESIGN: Retrospective case series. METHODS: Medical records, post-surgical racing performance and outcome of 26 horses with propagating fractures of the medial and/or lateral condyle of McIII/MtIII were reviewed. Medical information included were age, breed, sex, physical examination at admission, circumstances of fracture, radiographic evaluation, anaesthesia and recovery records, surgical and post-operative management, as well as complications. Outcome included racing data and information from telephone interviews. RESULTS: Twenty-six horses (9 Standardbreds and 17 Thoroughbreds) were admitted with a long condylar fracture of the McIII/MtIII. Fore- and hindlimbs were equally represented with the left hindlimb being more frequently involved. Most of the fractures had a spiralling component (76%) and four (15%) were comminuted. Fifteen (58%) horses raced post-surgery including nine Standardbreds (100%) and six Thoroughbreds (35%). Twelve of them were placed in at least one race and 11 won at least once. One horse sustained a severe complication in recovery. No significant difference was observed in the racing performances before and after surgery. MAIN LIMITATIONS: Follow-up method and duration were not standardised and there is a low number of cases with six surgeons. CONCLUSIONS: Long condylar fractures can be repaired using lag fashion technique combined with a half-limb or full-limb tight cast for recovery as a good surgical alternative. Similar results to plate fixation can be expected, with a return to racing of more than 50%, and the prognosis being even better for pacers.

A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa.

We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.

Identification and expression of macrophage migration inhibitory factor in Sarcoptes scabiei.

Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine produced by many mammalian tissues including skin. It is also found in many invertebrate parasites of mammals including ticks and may function to aid the parasite to evade the innate and adaptive immune responses in the host. In this study, the cDNA for a MIF gene was sequenced from Sarcoptes scabiei, the scabies mite, using RT-PCR and RACE molecular techniques. The resulting nucleotide sequence had a length of 405 base pairs and the putative amino acid sequences for the mite and tick (Dermacentor variabilis) proteins were identical. The initial steps for the project resulted in the production of expressed scabies mite cDNAs. A real time (qPCR) assay was performed with MIF from scabies mites and various tick species. Results show that mRNA encoding MIF homologues was three times more abundant in the mite samples when compared to RNA prepared from D. variabilis salivary glands and 1.3 times more abundant when compared with RNA prepared from D. variabilis midgut.

Hypoadiponectinemia is associated with valvular inflammation and faster disease progression in patients with aortic stenosis.

OBJECTIVES: Adiponectin is a protein secreted by adipocytes which has anti-inflammatory properties. The objective of this study was to examine the relationship between adiponectinemia and the hemodynamic progression of aortic stenosis (AS) as well as the degree of inflammation in the valve explanted at the time of aortic valve replacement (AVR). METHODS: The plasma level of adiponectin was measured in 122 patients undergoing AVR. The explanted aortic valves were analyzed and the density of leukocytes (CD45+), T cells (CD3+) and blood vessels (von Willebrand factor positive; vWF+) was documented. Also, a subset of patients (n = 67) had ≥2 echocardiographic studies separated by at least 6 months, thereby allowing assessment of the rate of progression of stenosis during the preoperative period. RESULTS: Patients with lower plasma levels of adiponectin (<5.4 µg/ml) had a faster progression rate of the mean transvalvular gradient before surgery than those with higher levels (9 ± 1 vs. 4 ± 1 mm Hg/year; p = 0.008). Moreover, these patients with hypoadiponectinemia had significantly more leukocytes (CD45+), T cells and blood vessels (vWF+) in their explanted valves compared to those with higher adiponectin levels. CONCLUSION: These findings support the concept that adiponectin may play a protective role against the inflammatory process and progression of calcific AS.

Acute small intestinal obstruction associated with Parascaris equorum infection in young horses: 25 cases (1985-2004).

AIMS: To retrospectively evaluate the medical and surgical records of horses with acute small intestinal obstructions associated with Parascaris equorum infection; to describe the gastrointestinal lesions; and to determine the outcome of cases with such lesions. METHODS: Records of 25 horses with acute small intestinal obstruction associated with P. equorum between 1985 and 2004 were reviewed to determine signalment, history, physical examination, surgical or post-mortem findings, and outcome. RESULTS: All horses except one were less than 12 months old. Standardbreds were over-represented in the population studied. Sixteen horses (72%) had been administered anthelmintics, including pyrantel (n=8), ivermectin (n=7), and trichlorphon (n=1), within 24 h prior to the onset of colic. Of the 25 cases reviewed, 16 had simple obstructive ascarid impactions (SOAIs), and nine had complicated obstructive ascarid impaction (COAI) including volvulus (n=6) or intussusception (n=3), both concurrent with ascarid impaction of the small intestine. Short-term survival (discharge from hospital) occurred in 79% of horses treated for SOAI, and was 64% for all horses. Long-term survival (>1 year) occurred in 33% of horses with SOAI, and the overall long-term survival was 27% for all horses. Formation of adhesions was the most frequent finding associated with death for horses that did not survive long-term. CONCLUSIONS AND CLINICAL RELEVANCE: The incidence of anthelmintic treatment within 24 h of the onset of colic in this study population (72%) was higher than that previously reported. Resistance of P. equorum to ivermectin recently reported in Ontario may be associated with increased ascarid burdens, predisposing horses to ascarid impaction. The long-term survival of these horses was better than that reported previously.

Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth.

REASONS FOR PERFORMING STUDY: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses. Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use. OBJECTIVES: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C. perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro. METHODS: Clostridium difficile toxins, C. perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests. Serial dilutions of smectite were tested for the ability to inhibit growth of C. perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C. perfringens in culture broth. RESULTS: Clostridium difficile toxins A and B, and C. perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16. Partial binding of C. difficile toxins occurred at dilutions up to 1:256 while partial binding of C. perfringens enterotoxin occurred up to a dilution of 1:128. Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32. No inhibition of growth of C. difficile or C. perfringens was present at any dilution, and there was no effect on the action of metronidazole. CONCLUSIONS: Di-tri-octahedral smectite possesses the ability to bind C. difficile toxins A and B, C. perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole. POTENTIAL RELEVANCE: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

Survival rates for horses undergoing stapled small intestinal anastomosis: 84 cases (1988-1997).

OBJECTIVE: To determine whether location and type of small intestinal anastomosis and other variables were associated with short- and long-term survival rates in horses undergoing stapled small intestinal anastomosis. DESIGN: Retrospective study. ANIMALS: 84 horses that underwent small intestinal anastomosis. PROCEDURE: Medical records from 1988 to 1997 were examined for horses that underwent stapled small intestinal anastomosis. Horses were allotted into 4 groups: jejunojejunostomy (n = 27), jejunoileostomy (11), jejunoileocecostomy with small intestinal resection (20), and jejunoileocecostomy without small intestinal resection (26). Survival rates and other variables were determined. RESULTS: Horses that underwent jejunoileocecostomy without resection had a significantly higher survival rate at all intervals than did horses in the other 3 groups. At 180 and 365 days after surgery, horses that underwent jejunojejunostomy had a significantly lower survival rate than those that underwent jejunoileocecostomy with intestinal resection. Horses that underwent jejunoileocecostomy with intestinal resection had a significantly higher survival rate at 1 year, compared with the combined jejunojejunostomy and jejunoileostomy groups. Preoperative heart rate was inversely associated with survival rate. Overall survival rates at discharge and 1 year were 81 and 56%, respectively. For horses that underwent small intestinal resection, survival rates at discharge and 1 year were 65 and 47%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Horses that underwent anastomosis of the small intestine to the cecum without resection had the highest survival rate, compared with horses that required intestinal resection. In some instances, resection and anastomosis involving the cecum had better prognosis than resections involving 2 segments of the small intestine.

Comparison of the in situ and in vivo intestinal disappearance of ruminally protected methionine.

We designed a switchback expriment to compare the intestinal disappearance of ruminally protected Met determined in situ or in vivo over three consecutive periods. Four nonlactating Holstein heifers (477+/-36 kg) with cannulae in the rumen, duodenum, and ileum were fed a diet based on timothy silage to meet requirements for maintenance (dry matter intake = 8.72+/-0.15 kg). A total of 16 bags, containing 1.5 g of ruminally protected Met, were incubated in the rumen (4.5 h) of each cow and transferred to an acid-pepsin solution to simulate the abomasum (2.5 h). Following each incubation, bags were recovered and three bags were dried at 55 degrees C and analyzed for Met. Remaining bags were introduced directly into the duodenal or ileal cannula for the in situ method, while for the in vivo method, the content of remaining bags was transferred into gelatin capsules before their introduction in the duodenal or ileal cannula. Spot samples of digesta were collected during a 96-h period, with Co-EDTA and Cr-mordanted fiber used as indigestible markers to estimate in vivo digestibility. The disappearance of Met in the small intestine determined in vivo tended to be higher than in situ (74.45 vs. 43.65+/-1.79%). Our results indicate that when used to assess intestinal availability of ruminally protected Met, the mobile nylon bag technique can underestimate the true bioavailability of Met.

Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation.

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

The presence of loose anagen hairs obtained by hair pull in the normal population.

To help determine the specificity of "loose anagen" (LA) hairs in Loose Anagen Syndrome, the presence or absence of LA hairs on a gentle but firm hair pull was evaluated in 110 normal subjects from a 0.5 to 83 y old. In children < or =10 y old, 61% had LA hairs on hair pull evaluation and 73% of all hairs obtained were LA hairs. In contrast, LA hairs were found in only two of 87 (2%) normal postpubescent subjects. The number of LA hairs was small in normal children (1-2 per hair pull) and a maximum of one out of every 6-7 hair pulls in adults, far less than that reported with Loose Anagen Syndrome. Although the mere presence of LA hairs on a hair pull test is thus not specific for LAS in children, the number per hair pull may have diagnostic significance. Correlation of these findings with the various hair disorder phenotypes currently termed Loose Anagen Syndrome will be important.

Interaction of transforming growth factor-beta-1 with alpha-2-macroglobulin from normal and inflamed equine joints.

Binding between equine plasma alpha-2-macroglobulin (alpha 2M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (125I-TGF-beta 1), recombinant human interleukin-1-beta (125I-IL-1 beta), and recombinant human tumor necrosis factor-alpha (125I-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha 2M to each cytokine was subjectively evaluated with autoradiography. Equine alpha 2M bound 125I-TGF-beta 1. However, poor or no binding was observed between alpha 2M and either of 125I-rhTNF-alpha or 125I-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with 125I-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha 2M were also quantified by densitometry of PAGE gels. Native alpha 2M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha 2M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, 125I-TGF-beta 1 bound predominantly to alpha 2M, and preferentially to the activated form of alpha 2M. In synovial fluid, the amount of 125I-TGF-beta 1 binding was proportional to the quantity of alpha 2M present. These results indicate that: 1) equine alpha 2M binds TGF-beta 1; 2) the native form of alpha 2M is present in both equine plasma and synovial fluid, and 3) alpha 2M is a major binding protein for TGF-beta 1 in equine synovial fluid. Therefore, alpha 2M may play a role in regulating this mediator of inflammation in equine joints.

Evaluation of plasma alpha-2-macroglobulin and interactions with tumour necrosis factor-alpha in horses with endotoxemic signs.

The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses.

Histamine synthesis inhibition reduces light-induced phase shifts of circadian rhythms.

Circadian rhythms are generated by the hypothalamic suprachiasmatic nuclei, a site of dense histaminergic innervation. Histamine can phase shift circadian rhythms in a manner similar to light. In this experiment, we administered alpha-fluoromethylhistidine (FMH), an inhibitor of histamine synthesis, prior to a light pulse in hamsters housed under constant darkness. Photic phase shifts in wheel-running rhythms were significantly attenuated by FMH pretreatment. These results suggest that histamine may modulate photic input to the circadian clock.

Histamine phase shifts the circadian clock in a manner similar to light.

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) receives a dense input from histamine-containing neurons in the posterior basal hypothalamus. We applied histamine to SCN tissue in vitro and measured the subsequent rhythm in firing rate. Histamine caused a phase delay in the early subjective night and a phase advance in the late subjective night. The similarity of histamine- and photic-induced phase shifts indicates that histamine may play a role in the modulation of circadian clock photic input.