Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro .
Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.
Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.
Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro , in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.     

Entérite nécrotique chez le poulet de gril I. Aspect clinico-pathologique

This study represents an analysis of the principal clinical factors and pathological lesions of 150 cases of necrotic enteritis encountered during 1968 and 1969 in Quebec. Following multiple investigations no common factor was observed which might have explained the pathogenesis of the condition which was observed during every month of the year, especially from May to November. Birds two to four weeks of age were the most susceptible. The main lesion was a fibrino-necrotic enteritis always localized in the small intestine, either in its entire length or just a segment and characterized by the disappearance of the surface epithelium and necrosis of the villi. The lumen was filled with desquamated epithelial cells and bacteria. Foci of coagulation necrosis were observed in the liver and foci of nephrosis in the kidneys. A Gram+ bacillus, strictly anaerobic, was always isolated from the viscera.     

Comparison of serological techniques to measure antibody to Pasteurella haemolytica A1.

Analysis of 45 sera was performed employing five techniques which are currently in use in three laboratories to measure anti-Pasteurella haemolytica antibodies. The enzyme linked immunosorbent assay, passive hemagglutination, complement fixation and direct and indirect bacterial agglutination assays were employed and a relationship between tests in the measurement of anti-P. haemolytica antibodies was demonstrated. Regression analysis together with prediction and confidence intervals were tabulated also. The conclusion drawn from statistical analysis was that all five tests are similar in their ability to detect immune responses (antibody and antigen(s) interactions) to Pasteurella haemolytica.     

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.     

Quantification of Fusarium solani f. sp. phaseoli in Mycorrhizal Bean Plants and Surrounding Mycorrhizosphere Soil Using Real-Time Polymerase Chain Reaction and Direct Isolations on Selective Media

ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.     

Alteration of virulence factors and rearrangement of pAsa5 plasmid caused by the growth of Aeromonas salmonicida in stressful conditions

Aeromonas salmonicida, a fish pathogen, is the causative agent of furunculosis. It was already shown that growing this bacterium in stressful conditions such as temperature above 22°C might lead to virulence attenuation. Unfortunately, many veterinary microbiology services and reference centers still routinely cultivate A. salmonicida at 25°C. Here we tested the presence of virulence factors by growth on specific medium as well as the integrity of the pAsa5 plasmid, which bears an important virulence factor, the type III secretion system (TTSS), by PCR analysis in twenty strains, most of which were grown at 25°C in their laboratory of origin. The analysis revealed that strains, which encountered the more stressful growth conditions displayed the most frequent absence of A-layer protein and secreted proteolytic activity. Moreover, many strains had lost parts of the pAsa5 plasmid in which the TTSS region was almost always affected. To confirm the effect of stressful growth conditions on the plasmid, three strains with an intact pAsa5 were cultured at 25°C for two weeks. A low but significant fraction of the tested colonies displayed pAsa5 rearrangements. The rearrangement always affected the TTSS region and led to a loss of virulence in the Dictyostelium discoideum co-culture assay. These results demonstrate that the instability of pAsa5 did not lead to its complete loss as previously proposed but to a more complex rearrangement phenomenon and emphasizes the necessity to grow A. salmonicida in appropriate conditions to preserve the complete virulence of the bacterium.