Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity

OBJECTIVES: To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. STUDY DESIGN: In vitro experimental study. ANIMALS: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). METHODS: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). RESULTS: Initial MSC isolates had a lag phase with a significantly longer CD time (DT=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages. CONCLUSIONS: The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species. CLINICAL RELEVANCE: The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.     

Validity and reliability of the Block98 food-frequency questionnaire in a sample of Canadian women

OBJECTIVE: To assess the validity and reliability of the most recent adaptation of Block's full-diet food-frequency questionnaire (FFQ) among a sample of Canadian women. DESIGN: Participants completed a self-administered FFQ (FFQ1), two unannounced 24-hour recalls (weekday and weekend) and a second FFQ (FFQ2) between October 2003 and February 2004. FFQs and recalls were analysed for 32 nutrients using Block Dietary Data Systems and the University of Minnesota's Nutrient Data System. Mean and median intakes were computed, along with crude and deattenuated Pearson correlation coefficients between FFQ1 and the average of two recalls (validity) and between FFQ1 and FFQ2 (reliability). SETTING: Ontario, Canada. SUBJECTS: A random population-based sample (n = 166) of women aged 25 to 74 years. RESULTS: One hundred and fifteen (69%) women completed FFQ1, 96 completed FFQ1 and both recalls, and 93 completed both FFQs, about 56 days apart. Mean intakes were similar for most nutrients. FFQ reliability was high, with Pearson correlation coefficients having a median of 0.75, ranging from 0.57 to 0.90 (macronutrients) and from 0.65 to 0.88 (micronutrients from supplements and food). FFQ validity was moderate to high, with deattenuated Pearson correlation coefficients having a median of 0.59, ranging from 0.11 to 0.73 (macronutrients) and from 0.50 to 0.76 (micronutrients from supplements and food). Our micronutrient correlations were similar to or higher than those of other studies that included supplements. Two correlations <0.40 were associated with fats. CONCLUSIONS: The validity and reliability of this full-diet version of the Block FFQ were moderate to high, supporting its use in future studies among Canadian women.     

Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.     

Western corn rootworm egg hatch and larval development under constant and varying temperatures

Predicting western corn rootworm (Diabrotica virgifera virgifera LeConte; WCR) development in the field depends on models that use experimentally determined degree-days (°D). For constant temperature regimes, this temperature sum can be reliably used to predict hatch and development of WCR larvae. In the first experiment in climate cabinets, we evaluated the effects of varying day–night (4 and 6 °C difference) temperature regimes compared to equivalent constant temperature regimes on hatch, development, and recovery of WCR larvae from a non-diapausing population. Relative to constant diurnal temperatures, varying day–night temperatures resulted in earlier larval hatch and accelerated larval development (especially when day–night temperatures differed by 6 °C) due to direct temperature effects (i.e., the Kaufmann-effect) and to enhanced plant growth. For WCR eggs, the temperature sums needed for hatch in the field are overestimated when they are determined by models based on constant experimental temperatures. Recovery of larvae from soil was not affected by temperature, but was positively associated with plant height. In a second experiment we evaluated whether the found effect of varying temperature ranges on the acceleration of larval hatch is also influenced by the level of these varying temperatures. Initial hatch started earlier by varying diurnal temperatures only under a low-temperature regime (14 ± 4 °C). For herbivorous insects like WCR, plant growth effects induced by varying temperature regimes may result in increased variation in temperature-based developmental parameters. Consideration of these effects will improve models that predict WCR hatching patterns and will improve the development and timing of control strategies.     

Comparison of Different Sampling Strategies and Laboratory Methods for the Detection of C. jejuni and C. coli from Broiler Flocks at Primary Production

Summary The objective of the study was to evaluate the performance of different combinations of sample type, transport medium and culture methods for the recovery of Campylobacter jejuni and C. coli from broiler flocks at primary production. Boot swabs moistened with one of four different transport media [maximum recovery diluent (n = 120), Exeter broth (EX) (n = 120), buffered peptone water (n = 120) and modified semi‐solid Cary‐Blair (n = 120)], caecal samples (n = 40) and faecal samples (n = 120) from 40 broiler flocks were compared and sensitivity estimates obtained using a Bayesian model. Samples were cultured onto mCCDA before and after enrichment in EX and incubated microaerobically at 41.5°C. Campylobacter suspect colonies were identified to the species level by multiplex PCR. Results from the Bayesian model indicated that boot swabs after enrichment had higher sensitivity (90–94%) than caecal contents before or after enrichment (84% and 89%, respectively) and faecal samples after enrichment (82%) for the detection of Campylobacter spp., although these differences were not statistically significant. Enrichment significantly increased the sensitivity of boot swab and caecal samples for detection of Campylobacter spp. and C. jejuni, respectively. However, the enrichment of caecal samples resulted in a significant decrease in the sensitivity of these samples for detection of C. coli. There was much greater variation in the sensitivity estimates of the methods for detecting C. coli than for C. jejuni, and the ranking of methods was different between the two species. Boot swabs gave the best sensitivity values for detection of C. jejuni, and enrichment culture of faecal samples was the most sensitive method for detection of C. coli.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

Salmonella enterica infections in Spanish swine fattening units

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella-positive sample (95% CI: 37-49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2-13.8%). The apparent herd prevalence of salmonellosis was similar among multi-site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi-resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively.