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.

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.