Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions.

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed.

Bilateral orbital and nasal aspergillosis in a cat.

A 12-year-old, 4 kg, castrated male Persian cat was referred with a 2-month history of sneezing and bilateral mucopurulent nasal discharge. Rhinoscopically acquired nasal biopsies at this time revealed bilateral lymphoplasmacytic rhinitis. A tapering dose of oral prednisone caused the complete remission of the clinical signs, but 2 months after discontinuation of the therapy, the rhinitis recurred and the OD became exophthalmic. Computed tomography showed a soft tissue mass in both sides of the nasal cavity, both frontal sinuses, the right orbit, and to a lesser extent the left orbit. A fine needle aspirate of the right orbit revealed pyogranulomatous inflammation and Aspergillus spp. hyphae. Repeat nasal biopsy demonstrated multi-focal necrosis and a mixed inflammatory cell process which now included macrophages and scattered septate fungal hyphae. A few days later the cat became bilaterally blind and a contrast enhancing lesion involving the optic chiasm was found on magnetic resonance imaging. Despite a poor prognosis, therapy consisted of exenteration of the right orbit and trephination of both frontal sinuses before the planned initiation of medical antifungal therapy. Unfortunately, the cat died of cardiac arrest intraoperatively. Aspergillus fumigatus was cultured from both orbits at necropsy. Orbital aspergillosis has been rarely reported in cats and its relationship with lymphoplasmacytic rhinitis is unclear. In this patient lymphoplasmacytic rhinitis or previous antibiotic/corticosteroid therapy may have allowed secondary fungal invasion of the nasal mucosa and subsequently both orbits and the brain. Alternatively, Aspergillus infection may have preceded the lymphoplasmacytic rhinitis.

Canine aural cholesteatoma: a histological and immunohistochemical study.

Canine aural cholesteatoma is an epidermoid cyst that forms in the middle ear cavity as a rare complication of otitis media but the aetiopathogenesis remains controversial. In the present study, 13 cases of canine aural cholesteatoma were investigated histologically and immunohistochemically and compared with cases of chronic otitis. The immunohistochemical investigation was performed using the following monoclonal antibodies: anti-cytokeratins (CK) 14, 16, 8/18, and 19, and anti-Ki67. The proliferative indexes (PIs) of cholesteatomata and otitis epithelium were calculated as the percentage of Ki67 positive nuclei/total nuclei. Histologically, the cholesteatomata were composed of a hyperplastic, hyperkeratotic epithelium (matrix) resting on a fibrous perimatrix, infiltrated by inflammatory cells and devoid of cutaneous adnexa. Immunohistochemically, the cholesteatoma epithelium was CK14- and CK16-positive, and CK8/18- and CK19-negative. A similar pattern of CK expression was found in otitis externa. In otitis media, ciliated epithelium stained CK8/18- and CK19-positive in all layers, CK14-positive in the basal layers, and CK16-negative. The mean PIs in cholesteatomata and otitides were 18.8 and 17.8, respectively. The immunohistochemical pattern of CK expression in cholesteatomata, when compared with chronic otitis, was suggestive of hyperproliferative epithelium, but its origin could not be demonstrated. Comparable PI values were obtained in cholesteatoma and in chronic otitis, which confirmed that Ki67 is a valuable indicator of a hyperproliferative state, but not a predictor of aggressiveness.