Analysis of the gacS-gacA regulatory genes of spontaneous mutants of Pseudomonas fluorescens biocontrol strain PfG32R

Pseudomonas fluorescens strain PfG32R, a potential biocontrol agent against soilborne pathogens, frequently loses its antifungal activity and ability to produce enzymes. To characterize genetically the instability of these bacterial functions, we analyzed gacS and gacA genes of PfG32R and three spontaneous mutants of PfG32R (NR1, NR9, and ASW6), which had lost their ability to produce proteases and antifungal activities. The gacS and gacA sequences of PfG32R had 77%–89% and 78%–87% homology, respectively, with several known gacS and gacA homologues. All three spontaneous mutants were subjected to complementation analysis. Introduction of clones containing an intact gacS of PfG32R and another P. fluorescens strain Pf-5 as well as strain tea632 of P. syringae pv. theae complemented all three mutants restoring protease and antifungal activities, indicating a mutation in gacS. In sequencing analysis, the mutants had a deletion or change in amino acids in the conserved sensor kinase domains of GacS. The three mutants maintained both their antibacterial activity against Ralstonia solanacearum and Clavibacter michiganensis ssp. michiganensis and siderophore production, indicating that they are not controlled by the GacS/GacA system. The sequences reported in this article have been deposited in the DDBJ database under accession numbers AB219364 and AB219365.     

Polypoid eosinophilic cystitis with pseudosarcomatous proliferative tissue in a dog

A dog presented with hematuria, and two small polypoid masses were detected in the urinary bladder. Histopathologically, the masses were located in the mucosal or submucosal layer. That tissue consisted of a random proliferation of spindle-shaped, round and pleomorphic cells with single or multiple large atypical nuclei and abundant cytoplasm, and eosinophil infiltration. These large cells were confirmed by immunohistochemical staining as fibroblasts, myofibroblasts and macrophages. Mitotic figure was rarely seen. These masses were diagnosed as eosinophilic polypoid cystitis with pseudosarcomatous proliferative tissue, since they consisted of a wide variety of cells and showed low growth activity.     

Isolation of Staphylococcus species from the tonsils of healthy cattle and phage patterns of isolates

Staphylococci were found in the tonsils of 121 (75.2%) of 161 cattle. There were 15 different species, 10 belonging to novobiocin-sensitive species. The most predominant species was S. simulans (79.3% of the 121 carriers), followed by S. aureus (20.7%), S. chromogenes (10.7%) and S. epidermidis (8.3%). The other 11 species were present in 0.8 to 5.8%. Twenty-six unidentifiable isolates were isolated from 26 (21.5%) carriers. Sixty-two (51.2%) of the 121 carriers yielded two to five Staphylococcus species together while only one species could be found in each of the other 59 (48.8%). Combinations of S. simulans and other species were most frequently encountered in 50 (41.3%) of the 121 carriers. Twenty-four (96.0%) out of 25 S. aureus isolates, 3 (42.9%) of 7 S. hyicus isolates and 45 (25.4%) of 177 coagulase-negative staphylococci (13 species and unidentifiable isolates) isolates were phage typable. Most of S. aureus isolates were lysed by bovine phages 119 (n = 16) or 116 (n = 5). Thirty-three (25.4%) of 45 coagulase-negative staphylococci typable isolates with Pulverer's phage set showed the phage pattern ph5/ph9/ph10/ph12/ph13/U4/U14/U16/++ +U20/U46. The tonsils of cattle thus appear to be a suitable environment for Staphylococcus species, particularly novobiocin-sensitive species.     

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.     

Prognostic significance of surgical margin, Ki-67 and cyclin D1 protein expression in grade II canine cutaneous mast cell tumor

The prognosis for canine cutaneous mast cell tumor (CCMT) is thought to be correlated with histopathological grading. However, the wide variety of histopathologic types of grade II is one of the most troublesome and difficult points for prognosis. The objective of this study is to determine the prognostic value of surgical margin, ki-67 and cyclin D1 protein expression in grade II tumor. Surgically resected specimens of solitary grade II CCMT from 48 dogs with follow-up periods over 360 days (median was 1080 days) were used in this study. The expression of cyclin D1 and ki-67 proteins was determined by morphometrically using slides stained immunocytochemically, and the correlations among the results, survival rate, and recurrence and/or metastasis rate of each dog were analyzed statistically. The recurrence and/or metastasis and mortality rate in the incomplete surgical excision group within 30 months postoperatively were higher than that of the complete surgical excision group. In the incomplete surgical excision group, dogs with low positive staining of ki-67 had a significantly better survival, but the recurrence and metastasis rate and ki-67 positivity failed to show a significant correlation. Only a small number of cases showed cyclin D1-positive tumor cells, but most of them had a poor outcome with a high recurrence rate. In grade II CCMT, incomplete excision induced a relatively high metastasis rate and poor prognosis. Ki-67 positivity is a marker for the estimation of overall survival in incomplete surgical excision cases. Cyclin D1 positivity was low and may not have a prognostic role.     

Three dogs under 2 years of age with heartworm caval syndrome

Heartworm caval syndrome (CS) occurred in three dogs under 2 years of age. A 23-month-old dog was recovered by surgical and medical treatments, but the other 2 dogs (15 and 21 months old) died. Necropsy demonstrated 12 heartworms in the 15-month-old dog and 8 worms in the 21-month-old dog. Histopathologically, pulmonary arterial embolism caused by dead worms and thrombi were observed in these cases. The findings suggested that CS could develop regardless of canine age and worm burden if pulmonary arterial embolism related to worm death or thrombus formation were induced.