E. coli 1303 was isolated from clinical cases; Inoculum Dose: Bovine mastitis isolate E. coli 1303 belongs to the major E. coli phylogenetic group A (E. coli collection of reference strains, ECOR-A). According to a multiplex PCR-based screening for virulence-associated genes of pathogenic E. coli, this strain does not represent an extraintestinal or intestinal pathogenic E. coli isolate. Only the genes coding for type 1 fimbriae, F17 fimbriae, antigen 43, the ferric citrate siderophore system and the EAST1 toxin could be detected. Bacteria were kept cryo-conserved (Mikrobank-system CryobankTM, Mast Diagnostika, Reinfeld, Germany) for subsequent infections. Bacteria were plated on Columbia sheep blood agar and incubated (37°C) for 24h. A few colonies were transferred to a tube of brain-heart infusion broth (Oxoid, Wesel, Germany) and incubated for 6h (37°C); then a 100-μl sample was transferred to a tube of trypticase soy broth (9.9ml, Oxoid, Wesel, Germany). Serial dilutions were made after 18h to prepare the desired inoculum dose of 500CFU/2ml 0.9% sterile, pyrogen free saline. The inoculum dose was plated for control and ranged from 421-613CFU/2ml. Inoculum was administered intracisternally in one quarter through the teat canal. Cows were slaughtered after 24h. Mammary gland tissue was isolated from quarters neighboring to infected quarters. Samples from untreated quarters from healthy animals served as controls.
Growth protocol
10 healthy, primiparous, midlactating German Holstein Frisian cows; no history of clinical mastitis; quarter milk samples less than 50,000 somatic cells/ml and free of mastitis pathogens; oestrus synchronized with Cloprostenol (Prostaglandin-F2alpha-analogue, Selectavet,Weyarn), before trial cows were entirely milked out with 20 I.U. Oxytocin i.v. (Veyx-Pharma, Schwarzenborn).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from bovine mammary gland tissue with the Trizol method according to the manufacturers protocol
Label
biotin
Label protocol
total RNA was transcribed and labeled with Ambion Kit MessageAmp II Biotin Enhanced according to the manufacturers protocol
Hybridization protocol
fragmented cRNA was hybridized to Affymetrix GeneChip Bovine Genome Arrays and washed and stained with Affymetrix Wash&Stain Kit on an Affymetrix Fluidics Station 450
