Investigation into in vitro and in vivo models using intestinal epithelial IPEC-J2 cells and Caenorhabditis elegans for selecting probiotic candidates to control porcine enterotoxigenic Escherichia coli.

AIMS: To identify a fast, economic and reliable method for preselecting lactic acid-producing bacterial (LAB) isolates to control enterotoxigenic Escherichia coli (ETEC). METHODS AND RESULTS: Two assays with porcine intestinal epithelial IPEC-J2 cells or Caenorhabditis elegans for selecting effective probiotic candidates were compared. Both assays were based on measuring death of cells or worms caused by ETEC strain JG280. Six of 13 LAB isolates showed ≥50% protection in each assay, among which only four isolates (≥50% protection) were consistently selected by both assays. Isolate CL9 (Lactobacillus reuteri) was further studied. It reduced gene expression of estA, estB and elt in JG280 in both assays. Furthermore, the isolate protected IPEC-J2 and C. elegans from cell and worm death caused by STa, STb or LT enterotoxin expressed in E. coli DH5α. CL9 also promoted host defensive responses by decreasing IL-8 and increasing IL-10 production in IPEC-J2 cells and expression of antimicrobial peptide genes clec-60, clec-85 in C. elegans. CONCLUSIONS: Caenorhabditis elegans is useful for preselecting probiotic candidates to control ETEC after initial screening with IPEC-J2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: A combination of IPEC-J2 cell and C. elegans assays can improve the effectiveness in preselecting probiotic candidates.

Toxicity assay for deoxynivalenol using Caenorhabditis elegans.

The progeny production and development rates of the nematode Caenorhabditis elegans when treated with deoxynivalenol (DON) were examined. Both purified DON and a crude extract from Fusarium graminearum cultured on rice were tested on C. elegans wild-type (Bristol N2) and a mutant strain (AU1). Significant effects (Tukey-HSD, p<0.05) on brood size and the rate of larval development from egg to adulthood were observed. Both N2 and AU1 strains showed lower rates of development and smaller brood sizes when exposed to purified DON at concentrations of 500 and 1000 µg ml(-1) When they were exposed to crude extract containing 250 µg ml(-1) DON, the inhibition of egg hatching and a greatly reduced development rate were observed. The results suggest that selection of a more sensitive C. elegans mutant strain could be used as a suitable animal model for conducting DON toxicity assays.

Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens.

AIMS: Determining the effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broilers. METHODS AND RESULTS: 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling, DNA sequencing and real-time quantitative PCR techniques were used. The richness of both ileal and caecal microbiota increased with chicken age. The microbiota from those birds of the same age demonstrated relatively similar PCR-DGGE profiles and tended to form closely related clusters in the relatedness analyses. Dietary treatment with bacitracin (50 mg kg(-1)) and access to range did not change the richness but altered the composition of the microbiota. The impact of bacitracin was particularly obvious in 3-day-old chicks. Lactobacilli were abundant in the caecal microbiota of 3-day-old chicks regardless of the dietary treatment with bacitracin. The access to range enriched Bifidobacterium in both the ileum and caeca. CONCLUSIONS: Bacitracin, bird age and access to range all influenced bacterial microbiota in the ileum and caeca of broilers, with bird age having the greatest apparent effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing useful information for the development of antibiotic replacement therapy for poultry production.

Efficacy and pharmacokinetics of bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle.

Bovine mastitis is an inflammation of the udder caused by microbial infection. Mastitis caused by Staphylococcus aureus is a major concern to the dairy industry due to its resistance to antibiotic treatment and its propensity to recur chronically. Growing concerns surrounding antibiotic resistance have spurred research into alternative treatment methods. The ability of lytic S. aureus bacteriophage K to eliminate bovine S. aureus intramammary infection during lactation was evaluated in a placebo-controlled, multisite trial. Twenty-four lactating Holstein cows with preexisting subclinical S. aureus mastitis were treated. Treatment consisted of 10-ml intramammary infusions of either 1.25 x 10(11) PFU of phage K or saline, administered once per day for 5 days. The cure rate was established by the assessment of four serial samples collected following treatment. The cure rate was 3 of 18 quarters (16.7%) in the phage-treated group, while none of the 20 saline-treated quarters were cured. This difference was not statistically significant. The effects of phage intramammary infusion on the bovine mammary gland were also studied. In healthy lactating cows, a single infusion of either filter-sterilized broth lysate or a CsCl gradient-purified phage preparation elicited a large increase in the milk somatic cell count. This response was not observed when phage was infused into quarters which were already infected with S. aureus. Phage-infused healthy quarters continued to shed viable bacteriophage into the milk for up to 36 h postinfusion. The phage concentration in the milk suggested that there was significant degradation or inactivation of the infused phage within the gland.

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K.

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.

Molecular typing and distribution of Staphylococcus aureus isolates in Eastern Canadian dairy herds.

Macrorestriction analysis of SmaI-digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288 Staphylococcus aureus isolates recovered from 58 Eastern Canadian dairy herds. In addition, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships, the PFGE types were assigned to six lineage groups, designated A through F. Of all of the isolates, ca. 93% were assigned to lineage groups A, D, or F. In 58.6% of herds, only a single PFGE type was recovered, while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the S. aureus isolates. Distribution of PFGE types and phage types was independent across regions and within herds.

Association of nuclear matrix proteins with cytoplasmic assembly sites of Tipula iridescent virus.

Intact viroplasmic centers were isolated from Estigmene acres cells infected with Tipula iridescent virus (TIV) by homogenization, followed by differential and discontinuous sucrose gradient centrifugation. Labeling of in situ and isolated viral assembly sites by two monoclonal antibodies raised against lymphocyte nuclear matrix proteins indicated a possible involvement of highly conserved nuclear proteins in the assembly and maturation of virions, as well as in maintaining the integrity of membrane-free viroplasmic centres. Electron microscopy and immunofluorescence of intact and fractionated E. acrea cells at different times postinfection showed no evidence of cytoskeleton involvement in the formation and maintenance of TIV viroplasmic centers.