In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either approximately 4.0 × 10(5) colony-forming units (CFU) or approximately 1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.

Objective—To assess expression pattern and subcellular compartmentalization of 5-lipoxygenase in cutaneous, UV radiation–induced, and oral squamous cell carcinomas (SCCs) in cats and determine the effects of cyclooxygenase or 5-lipoxygenase inhibition on proliferation or apoptosis in a feline oral squamous cell carcinoma (SCCF1) cell line. Sample—60 archived paraffin-embedded samples of SCCs from 60 cats and SCCF1 cells. Procedures—Retrospective immunohistochemical analysis of the archived samples of SCCs (20 cutaneous, 20 UV radiation–induced, and 20 oral tumors) was performed. Cell culture proliferation assays involving SCCF1 cells were performed, and tepoxalin-induced apoptosis and signaling were examined via western blotting and annexin V staining. Results—Immunohistochemically, staining for 5-lipoxygenase was most frequently of greatest intensity in oral SCCs, whereas staining of cutaneous and UV radiation–induced lesions had less consistent 5-lipoxygenase expression. Exposure of SCCF1 cells to the 5-lipoxygenase inhibitor tepoxalin resulted in apoptosis; the effect appeared to be mediated via alteration of cell signaling rather than via suppression of lipid mediators that are typically produced as a result of 5-lipoxygenase activity. Conclusions and Clinical Relevance—In cats, expression of 5-lipoxygenase in SCCs appeared to differ depending on tumor location. The influence of tepoxalin-induced 5-lipoxygenase inhibition on a poxygenase–expressing cell line coupled with the notable expression of 5-lipoxygenase in oral SCCs suggested that 5-lipoxygenase inhibition may have therapeutic benefits in affected cats. Although the safety of tepoxalin in cats has yet to be investigated, 5-lipoxygenase inhibitors should be evaluated for use as a potential treatment for SCCs in that species.

Objective—To characterize and compare the urine protein content in cats without urinary tract disease and cats with idiopathic cystitis (IdC), bacterial urinary tract infection (UTI), or urolithiasis. Animals—Control cats (n = 18) and cats with IdC (18), UTI (12), and urolithiasis (12) from which urine samples were obtained and 2 cats with obstructive IdC and 4 additional control cats from which postmortem urinary bladder biopsy specimens were obtained. Procedures—Protein contents in urine samples obtained via cystocentesis or catheterization were measured via the Bradford method. Urine proteins were separated by means of 1-dimensional gel electrophoresis. Evaluation of fibronectin content was performed via western blotting and immunohistochemical analysis. Urinary bladder biopsy specimens were examined histologically and analyzed immunohistochemically for fibronectin. Results—Urine fibronectin content was significantly greater in cats with IdC, compared with control cat findings. Urine fibronectin contents did not differ significantly among controls and cats with UTI or urolithiasis. Histologic examination of bladder biopsy specimens obtained from 2 cats with obstructive IdC revealed destruction of the urothelial lining of the urinary bladder and severe fibrosis; immunohistochemical analysis revealed few fluorescence signals for fibronectin, unlike findings in control bladder biopsy specimens. Conclusions and Clinical Relevance—Results indicated that urine fibronectin content in cats with IdC was greater than that in controls, cats with UTI, or cats with urolithiasis. In cats with IdC, increased permeability of damaged urothelium may result in detachment and leakage of fibronectin into urine. Urine fibronectin might serve as a biomarker for diagnosis of IdC in cats.

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

Objective—To examine the changes in monocarboxylate transporter (MCT) 1 and MCT4 content and in indicators of energy metabolism in the gluteus medius muscle (GMM) of Thoroughbreds during growth. Animals—6 Thoroughbreds (3 males and 3 females). Procedures—Samples of GMM were obtained when horses were 2, 6, 12, and 24 months old. Muscle proteins were separated via SDS-PAGE; amounts of MCT1 and MCT4 and peroxisome proliferator-activated receptor-γ coactivator-1α content were determined by use of western blotting. Muscle activities of phosphofructokinase and citrate synthase were measured biochemically; lactate dehydrogenase isoenzymes were separated by agarose gel electrophoresis and quantified. Results—Compared with findings when horses were 2 months old, MCT1 protein content in GMM samples obtained when the horses were 24 months old was significantly higher; however, MCT4 protein content remained unchanged throughout the study period. Peroxisome proliferator-activated receptor-γ coactivator-1α content was significantly increased at 24 months of age and citrate synthase activity was increased at 6 and 24 months of age, compared with findings at 2 months. Phosphofructokinase activity remained unaltered during growth. The percentage contributions of lactate dehydrogenase 1 and 2 isoenzymes to the total amount of all 5 isoenzymes at 12 and 24 months of age were significantly higher than those at 2 months of age. Conclusions and Clinical Relevance—Changes in protein contents of MCTs and the lactate dehydrogenase isoenzyme profile in GMM samples suggested that lactate usage capacity increases with growth and is accompanied by an increase in the oxidative capacity in Thoroughbreds.

Objective—To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells. Sample—Human adrenocortical carcinoma cells. Procedures—Melatonin plus ENL or END was added to cell culture medium along with cAMP (100μM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentration by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content. Results—The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concentrations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower. Conclusions and Clinical Relevance—The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism.

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

The present study investigated whether the 14-3-3 η and γ proteins, which are potent matrix metalloprotease (MMP) stimulators, are detectable in the synovial fluid of dogs with cranial cruciate ligament rupture (CCLR). Synovial fluid samples from 7 dogs with unilateral CCLR and control samples from 4 dogs without a history of any joint inflammation or any other abnormalities underwent Western blot analysis for the 14-3-3 η, γ, and σ proteins as well as MMP-1 and MMP-3. Craniocaudal and lateral radiographic projections of the stifle joint were evaluated for the presence and severity of 13 specific radiographic markers of osteoarthritis and graded numerically. The Spearman method was used to detect any correlation between the 14-3-3-η level in the synovial fluid and the radiograph-based grade. The η isoform was present only in the samples from the dogs with CCLR. The levels of 14-3-3-γ, MMP-1, and MMP-3 were significantly higher in the samples from the dogs with CCLR than in the control samples (P < 0.05). However, there was no significant difference between the CCLR and control samples in the level of the σ isoform. The Spearman method showed a significant correlation between the 14-3-3-η level in the synovial fluid and the presence of either patellar osteophytes or lateral or medial (or both) condylar periarticular osteophytes (P < 0.05). The MMP stimulatory effect of the 14-3-3 η and γ isoforms may be the reason for the high levels of MMP-1 and MMP-3 observed. Thus, 14-3-3 proteins, especially the η isoform, may be important markers of osteoarthritis caused by CCLR.