PCR analysis of bisulfite-modified DNA to investigate developmental variation in micropropagated potatoes (Solanum tuberosum L.)

Summary Bisulfite modification of DNA from potato leaves showing juvenile and mature leaf morphologies and from leaves of autotrophic and heterotrophic microplants showing different morphologies was carried out to deaminate cytosine residues while leaving 5-methylcytosine residues intact. RAPD analysis of the DNA was then performed using selective primers; firstly, with a 50–70% GC content and subsequently with a 50–70% AT content which contained CG or CA 3' ends. Three of the 4 primers used detected band polymorphisms in the juvenile versus mature leaves and all four primers detected band polymorphisms in the autotrophic·versus heterotrophic in vitro leaf samples. These results indicate differences in methylation in the different paired DNA samples. The hypothesised increase in band numbers in the relatively less mature samples was confirmed for both the in vivo and in vitro leaf samples; a higher number of bands was amplified from the DNA of the leaves from heterotrophic cultures compared with those from autotrophic culture when the results for all primer sets were grouped. however, this trend did not apply to the results from all the individual primers. The application of this approach to the evaluation of in vitro protocols for potato micropropagation is discussed.     

Evaluation of Lactobacillus pentosus WE7 for prevention of diarrhea in neonatal foals

OBJECTIVE: To evaluate the efficacy of Lactobacillus pentosus WE7, an equine-origin organism with potentially beneficial in vitro properties, as a probiotic for prevention of neonatal diarrhea in foals. DESIGN: Randomized controlled clinical trial. ANIMALS: 153 foals. PROCEDURE: Foals were enrolled at 24 to 48 hours of age and randomly assigned to treatment or control groups. The treatment group received approximately 2 x 10(11) CFU of freeze-dried L. pentosus WE7 orally once daily for 7 days, whereas the control group received a placebo. Clinical monitoring was performed for 14 days. RESULTS: Probiotic administration was significantly associated with development of signs of depression, anorexia, and colic and the need for veterinary examination and treatment. Probiotic-treated foals also had more days of diarrhea, compared with the control group, although not significantly. In a multivariate model, probiotic administration was significantly associated with development of diarrhea and diarrhea plus additional clinical abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of L. pentosus WE7 did not prevent diarrhea; rather, it was associated with development of diarrhea and, most importantly, development of diarrhea plus additional clinical abnormalities and the need for veterinary intervention. The promising in vitro properties of L. pentosus WE7 were not evident in vivo. Results raise concern about the variety of untested probiotic products that are commercially available. Safety and efficacy testing needs to be performed for all potential equine probiotics.     

Suspected Clostridium difficile-associated hemorrhagic diarrhea in a 1-week-old elk calf

Clostridium difficile-associated diarrhea was suspected in a 1-week-old elk (Cervus elaphus) calf. The isolation of a toxigenic strain of C. difficile from a diarrheic fecal sample, along with exclusion of other enteropathogens, formed the basis of this presumptive diagnosis. Further study is indicated to evaluate the role of C. difficile in neonatal diarrhea in elk.     

Antimicrobial susceptibility of hazard analysis critical control point Escherichia coli isolates from federally inspected beef processing plants in Alberta, Saskatchewan, and Ontario

A survey to estimate the prevalence of antimicrobial resistance in Escherichia coli was conducted in 7 Canadian federally inspected processing plants during 2001. Escherichia coli isolates were recovered during routine hazard analysis critical control point sampling from beef carcasses and trim and subsequently tested for their antimicrobial susceptibility by using susceptibility panels. Of the 2653 isolates analyzed, 68% were sensitive to all 18 antimicrobials tested. For 14 of the 18 antimicrobials evaluated, the percentage of resistant isolates was < or = 1. Twenty-five percent of the isolates were resistant to tetracycline, 9% to sulfamethoxazole, 7% to streptomycin, and 3% to ampicillin. Multiple resistance was found in 12% of the isolates, with 7% showing resistance to 2 antimicrobials, 2% to 3 antimicrobials, 2% to 4 antimicrobials, and 1% to 5 or more antimicrobials. Forty-five different antimicrobial resistance patterns were observed. The reasons for the development of the antimicrobial resistance were not investigated in this study. This study was useful as a pilot to help to develop a national antimicrobial resistance surveillance program in Canada. This study indicates that laboratory standardization is possible for consistent results across the country and that the indicator organism, E. coli, is fairly easy to obtain for surveillance but Salmonella are not, due to their low prevalence in beef.     

Expression of the uterine Mx protein in cyclic and pregnant cows,gilts, and mares

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.     

Presumptive primary cardiac lymphoma in a cat causing pericardial effusion

We report a case of primary cardiac lymphoma in a cat, causing pericardial effusion. A 13-year-old castrated male Himalayan cat was evaluated for chronic weight loss and radiographic finding of cardiomegaly. Pericardial effusion and a heart mass were detected via echocardiography. Pericardiocentesis and ultrasound-guided fine needle aspirate of the heart mass were performed under sedation. Antemortem diagnosis of cardiac lymphoma was made based on cytology of pericardial fluid. Based on physical examination, laboratory tests and abdominal radiographs, primary cardiac lymphoma was established as the presumptive clinical diagnosis. Treatment with chemotherapeutic agents was initiated.