Analysis of the immunomodulatory properties of mycobacterium cell wall fraction on the cytokine production of peripheral blood mononuclear cells of healthy dogs.

BACKGROUND: Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research. OBJECTIVE: To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines. ANIMALS: Eight healthy Labrador retrievers. MATERIALS AND METHODS: PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta. RESULTS: A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 µg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 µg/dL MCWF only at 72 h (p < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.

Evaluation of the impact of tromethamine edetate disodium dihydrate on antimicrobial susceptibility of Pseudomonas aeruginosa in biofilm in vitro.

BACKGROUND: Biofilm formation by Pseudomonas aeruginosa has been documented in canine otic isolates. An increase in minimal inhibitory concentration (MIC) for specific antibiotics has been noted for biofilm-embedded bacteria. Tromethamine edetate disodium dihydrate buffered to pH 8 with tromethamine hydrochloride and deionized water (Triz-EDTA(®)) has been documented to potentiate bactericidal activity when used in combination with topical antibiotics, but the impact on biofilm-embedded bacteria is unknown. HYPOTHESIS/OBJECTIVES: The objective of this study was to evaluate the impact of Triz-EDTA(®) use on in vitro antimicrobial susceptibility of biofilm-embedded P. aeruginosa. METHODS: Biofilm formation was documented using a microtitre plate assay. Broth microdilution was used to assess the MIC of neomycin, polymyxin B, enrofloxacin and gentamicin for the biofilm-embedded bacteria. The microtitre plate assay was again used to assess the MIC of neomycin, polymyxin B, enrofloxacin and gentamicin for biofilm-embedded bacteria with added Triz-EDTA(®). RESULTS: Thirty-one isolates from dogs with otitis were tested. Addition of Triz-EDTA(®) significantly reduced MICs for neomycin (P < 0.003) and gentamicin (P < 0.02) but not for polymyxin B (P = 0.3). Enrofloxacin MICs increased in the presence of Triz-EDTA (P < 0.036). CONCLUSIONS AND CLINICAL IMPORTANCE: Triz-EDTA(®) may be a useful adjunctive treatment for chronic cases of Pseudomonas otitis where biofilms may have developed, if gentamicin or neomycin is to be used as a topical treatment. In vivo study is required to confirm this effect.

Evaluation of biofilm production by Pseudomonas aeruginosa from canine ears and the impact of biofilm on antimicrobial susceptibility in vitro.

BACKGROUND: Pseudomonas aeruginosa is a common cause of canine otitis; P. aeruginosa biofilm formation has been documented in human medicine, but the role of biofilms in canine disease is not well documented. Bacteria within biofilms can be more resistant to antibiotics compared with their planktonic form; therefore, understanding the biofilm-forming capacity of isolates and their susceptibility to antimicrobials is important when developing treatment regimens. HYPOTHESIS/OBJECTIVES: To evaluate the biofilm-forming capacity of canine otic isolates of P. aeruginosa and to compare the minimal inhibitory concentration (MIC) of the planktonic versus biofilm-embedded bacteria. METHODS: Biofilm-forming ability was assessed using a microtitre plate assay. Broth microdilution was used to assess the MICs of neomycin, polymyxin B, enrofloxacin and gentamicin for the planktonic and biofilm-embedded bacteria. RESULTS: Eighty-three isolates from dogs with otitis were tested; 33 (40%) were classified as biofilm producers. Biofilm MICs for polymyxin B, neomycin, gentamicin and enrofloxacin were significantly higher than for the planktonic form (P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: Biofilm production by otitis isolates of P. aeruginosa is common and may play a role in the pathogenesis of disease. The MICs for biofilm-embedded bacteria differ from their planktonic counterparts, potentially leading to a lack of response to treatment. If polymyxin B, gentamicin, neomycin or enrofloxacin is to be used for topical treatment of a Pseudomonas otitis, the concentration of the medication should be increased, in particular if addressing chronic otitis, because biofilms may have developed.

Uveodermatologic syndrome in an Akita.

An 8-year-old, spayed, female Japanese Akita was presented for acute blindness, cloudy eyes, and squinting. A presumptive diagnosis of uveodermatologic syndrome was made. Therapy with oral prednisone, topical prednisolone, and oral azathioprine was successful in eliminating most of the clinical signs and the Akita now has complete restoration of vision.